Assays for fungal infection

ABSTRACT

Methods and kits are described for testing for the presence or absence of any fungus in a sample. Examples of fungi that can be detected include, but are not limited to, those belonging to the genera Candida, Aspergillus and Pneumocystis. The methods include obtaining a sample suspected of containing fungal nucleic acid, including at least one universal region of fungal nucleic acid, and testing for the presence or absence in the sample of the at least one universal region of fungal nucleic acid. Samples may be biological or non-biological.

INFORMATION ON RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/447,606, filed May 12, 2010, now U.S. Pat. No. 9,657,355, which isthe U.S. National Phase of International Application No.PCT/US2007/023043, filed Nov. 1, 2007, which claims priority to U.S.Provisional Application No. 60/968,413, filed Aug. 28, 2007, and to U.S.Provisional Application No. 60/864,146, filed Nov. 2, 2006, and to UKPatent Application No. 0716687.9, filed Aug. 28, 2007, and to UK PatentApplication No. 0621864.8, filed Nov. 2, 2006, all of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to detecting fungi in a sample. In particular, theinvention relates to the rapid detection of any fungus in a sample andthe rapid detection of a particular genus or species of fungus in asample. The invention also generally relates to fungal identification.Methods and kits are also provided for testing for the presence orabsence of a fungus in a sample. Examples of fungi detected include, butare not limited to, those belonging to the genera Candida, Aspergillusand Pneumocystis.

BACKGROUND OF THE INVENTION

Fungal infections are a significant cause of morbidity and mortality ina variety of severely ill patients. For instance, fungi are able tocause superficial and often fatal disseminated infections inimmunocompromised patients. Systemic fungal infections causeapproximately 25% of infection-related deaths in leukaemics and 5-10% ofdeaths in patients undergoing lung, pancreas or liver transplantation.Acquired fungal sepsis is also known to occur in up to 13% of very lowbirth-weight infants.

Members of the Candida genus are responsible for most of the fungalinfections in humans. They are the fourth most common cause ofnosocomial bloodstream infections. However, other fungal species arealso responsible for infections in humans. The members of theAspergillus genus are the second most common cause of fungal infectionsbehind the members of the Candida genus. However, other genera,including Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus,Mucor and Absidia, can be responsible for disseminated infections inhumans.

The impact of a fungal infection is often exacerbated by a failure torapidly diagnose and effectively treat the infection. Numerous studieshave shown that a delay in appropriate therapy is associated withincreased morbidity and mortality. At present, clinical methods ofdetecting the presence of a fungus in a patient are unreliable and timeconsuming. For instance, the detection of fungal infections is usuallycarried out by blood culture, which takes up to 1 to 2 days to performand often provides false negative results. In addition, some fungi, suchas members of the Pneumocystis genus, cannot be cultured easily.

The lack of rapid diagnostic tests for particular fungal species is oneof the major impediments to successful management of infected patients.Different species of fungus, even in the same genus, vary in theirsusceptibilities to the common anti-fungal agents. Some species evendisplay resistance to some agents. The identification of the specificfungal species causing an infection can be even more time consuming thatsimply detecting the presence of a fungus and this can often furtherdelay effective treatment of the infection.

It is also important that various non-biological samples, such assurgical fluids and drinking water, are known to be fungus free.However, there is currently a lack of rapid tests for the presence of afungus or for particular fungal genera or species in non-biologicalsamples.

SUMMARY OF THE INVENTION

The inventors have shown that the presence or absence of any fungus in asample can be rapidly and reliably determined using DNA analysis. Thisis referred to herein as panfungal detection. In particular, theinventors have shown for the first time that panfungal detection can becarried out on non-biological samples. The inventors have alsoidentified a novel region of fungal DNA that can be used for panfungaldetection.

The inventors have also identified novel regions of DNA that arespecific to fungi belonging to the genera Candida, Aspergillus andPneumocystis and can therefore be used to detected fungi belonging toeach of these genera. The inventors have further developed novel probesand primers that can be used to detect these regions either individuallyor simultaneously in a multiplex reaction.

The inventors have also developed novel primers and probes for the rapiddetection of fungi belonging to the species Candida tropicalis, Candidaparapsilosis, Candida albicans, Candida glabrata and Candida krusei.This allows all of these species to be identified simultaneously in amultiplex reaction using only one pair of primers. The inventors havealso developed a new internal PCR amplification control.

The invention therefore concerns the rapid detection of any fungus in asample. The invention also concerns the rapid detection of fungi of thegenus Candida, Aspergillus or Pneumocystis in a sample. The inventionfurther concerns the rapid detection of fungi of the species Candidatropicalis, Candida parapsilosis, Candida albicans, Candida glabrata orCandida krusei in a sample. The invention means that DNA analysis can beused to rapidly and reliably determine the presence of or the genus orspecies of a fungus in a sample. Accordingly, the invention provides amethod for the rapid detection of the presence or absence of any fungusin a sample, comprising detecting the presence or absence in the sampleof at least one universal region of fungal DNA. The invention furtherprovides:

-   -   a method for the rapid detection of the presence or absence of a        fungus belonging to the genus Candida in a sample, comprising        detecting the presence or absence in the sample of a region of        fungal DNA that shares at least 80% homology with SEQ ID NO: 5;    -   a method for the rapid detection of the presence or absence of a        fungus belonging to the genus Aspergillus in a sample,        comprising detecting the presence or absence in the sample of a        region of fungal DNA that shares at least 80% homology with SEQ        ID NO: 14;    -   a method for the rapid detection of the presence or absence of a        fungus belonging to the genus Pneumocystis in a sample obtained        from a human, comprising detecting the presence or absence in        the sample of a region of fungal DNA that shares at least 80%        homology with SEQ ID NO: 18;    -   a method for the rapid detection of the presence or absence of a        fungus belonging to the species Candida tropicalis in a sample,        comprising detecting the presence or absence in the sample of a        region of fungal DNA as shown in SEQ ID NO: 30 using the        molecular beacon probe shown in SEQ ID NO: 33;    -   a method for the rapid detection of the presence or absence of a        fungus belonging to the species Candida parapsilosis in a        sample, comprising detecting the presence or absence in the        sample of a region of fungal DNA as shown in SEQ ID NO: 34 using        the molecular beacon probe shown in SEQ ID NO: 35;    -   a method for the rapid detection of the presence or absence of a        fungus belonging to the species Candida albicans in a sample,        comprising detecting the presence or absence in the sample of a        region of fungal DNA as shown in SEQ ID NO: 36 using the        molecular beacon probe shown in SEQ ID NO: 37;    -   a method for the rapid detection of the presence or absence of a        fungus belonging to the species Candida glabrata in a sample,        comprising detecting the presence or absence in the sample of a        region of fungal DNA as shown in SEQ ID NO: 38 using the        molecular beacon probe shown in SEQ ID NO: 39;    -   a method for the rapid detection of the presence or absence of a        fungus belonging to the species Candida krusei in a sample,        comprising detecting the presence or absence in the sample of a        region of fungal DNA as shown in SEQ ID NO: 40 using the        molecular beacon probe shown in SEQ ID NO: 43;    -   a kit for the rapid detection of the presence or absence of any        fungus in a sample, comprising the molecular beacon probe as        shown in SEQ ID NO: 4 and the primers shown in SEQ ID NOs: 2 and        3;    -   a kit for the rapid detection of the presence or absence of any        fungus in a sample, comprising (a) the molecular beacon probe as        shown in SEQ ID NO: 44; (b) one or both of the sense primers        shown in SEQ ID NOs: 48 and 50; and (b) one or both of the        antisense primers shown in SEQ ID NOs: 49 and 51;    -   a kit for the rapid detection of the presence or absence of any        fungus in a sample, comprising (a) the molecular beacon probe as        shown in SEQ ID NO: 45; (b) one or both of the sense primers        shown in SEQ ID NOs: 48 and 50; and (b) one or both of the        antisense primers shown in SEQ ID NOs: 49 and 51;    -   a kit for the rapid detection of the presence or absence of a        fungus belonging to the genus Candida in a sample, comprising        the molecular beacon probe shown in SEQ ID NO: 8 and the primers        shown in SEQ ID NOs: 6 and 7;    -   a kit for the rapid detection of the presence or absence of a        fungus belonging to the genus Aspergillus in a sample,        comprising the molecular beacon probe shown in SEQ ID NO: 17 and        the primers shown in (a) SEQ ID NOs: 15 and 16 or (b) SEQ ID        NOs: 46 and 47; a kit for the rapid detection of the presence or        absence of a fungus belonging to the genus Pneumocystis in a        sample obtained from a human, comprising the molecular beacon        probe shown in SEQ ID NO: 21 and the primers shown in SEQ ID        NOs: 19 and 20; and a kit for the rapid detection of the        presence or absence of a fungus belonging to a particular        species of Candida in a sample, comprising the primers shown in        SEQ ID NOs: 31 and 32 or 41 and 42 and one or more of the        molecular beacon probes shown in SEQ ID NOs 33, 35, 37, 39 and        43.

In one aspect, methods are provided for testing for the presence orabsence of any fungus in a sample, comprising obtaining a samplesuspected of containing fungal nucleic acid, including at least oneuniversal region of fungal nucleic acid, and testing for the presence orabsence in the sample of the at least one universal region of fungalnucleic acid. In some embodiments, the nucleic acid comprises DNA, andin other embodiments, the nucleic acid comprises RNA. Samples may beobtained from a wide variety of biological and/or non-biologicalsources.

In some embodied methods the testing step includes contacting the samplewith an oligonucleotide probe comprising a nucleic acid capable ofhybridizing to the at least one universal region of fungal nucleic acidunder stringent conditions. The testing step may also include contactingthe sample with an oligonucleotide probe comprising a nucleic acidcapable of hybridizing to the at least one universal region of fungalnucleic acid under non-stringent conditions. In some embodiments, themethods further comprise amplifying the at least one universal region offungal nucleic acid. In methods in which the universal region of thefungal nucleic acid is amplified, the amplifying step may be carried outin the presence of one or more internal PCR amplification controls toensure appropriate amplification of any fungal nucleic acid present inthe sample. In such methods the one or more internal PCR amplificationcontrols may comprise a non-fungal sequence.

In another aspect, methods of testing for the presence or absence of afungus belonging to a genus such as Candida, Aspergillus, orPneumocystis in a sample are provided. Such methods may comprisingobtaining a sample suspected of containing fungal nucleic acid,including at least one region of fungal nucleic acid characteristic ofthe genus of interest, and testing for the presence or absence in thesample of the at least one region of fungal nucleic acid characteristicof the that genus. The testing step may include contacting the samplewith a probe. Such methods may further comprise amplifying the at leastone region of fungal nucleic acid characteristic of the genus bycontacting the sample with a pair of primers.

In yet another aspect, methods of testing for the presence or absence ofa fungus belonging to species such as Candida tropicalis, Candidaparapsilosis, Candida albicans, Candida glabrata, and/or Candida kruseiin a sample are provided. Such methods may comprise, obtaining a samplesuspected of containing fungal nucleic acid, including at least oneregion of fungal nucleic acid characteristic of the species of interest,and testing for the presence or absence in the sample of the at leastone region of fungal nucleic acid characteristic of the that species. Inthe above embodied methods in which the fungus belongs to a Candidaspecies, the methods may further comprise amplifying the at least oneregion of fungal nucleic acid characteristic of the Candida species bycontacting the sample with a pair of primers.

In another aspect, testing kits are provided for a number of fungalnucleic acids. In some embodiments, a panfungal nucleic acid testingkits are provided. In other embodiments, kits are provided for testingfor the presence or absence of at least one region of a fungal nucleicacid characteristic of the genus Aspergillus, of the genus Pneumocystis,or of a species of Candida in a sample. In another example, kits areprovided for simultaneously detecting panfungal, panCandida andpanAspergillus targets. Other kits comprise reagents for detectingpanAspergillus targets and Pneuomcytis jirovecii. Such kits may furthercomprise one or more internal PCT amplification controls.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a plot of the fluorescence released from the panfungalmolecular beacon (Table 1, Ib; SEQ ID NO: 4) as the temperature isincreased in the presence or absence of the target (melting curve). Thedotted line shows the results with the target. The continuous line showsthe results without the target.

FIG. 2 shows the detection of Candida (PanCan), Aspergillus (PanAsp) andfungal (PanFun) DNA together with an internal control (IC) in amultiplex reaction.

FIG. 3 shows the detection of Pneumocystis (PCP), Aspergillus (PanAsp)and any fungal (PanFun) DNA together with an internal control (IC) in amultiplex reaction.

FIG. 4 shows the detection of fungal DNA in various water samples from aHospital.

FIGS. 5A, 5B and 5C show a sequence alignment between the 18S region ofvarious members of the Candida genus. Ctrop is Candida tropicalis. Cparis Candida parapsilosis. Cdub is Candida dubliniensis. Calb is Candidaalbicans. Cguil is Candida guilliermondii. Clus is Candida lusitaniae.Cgla is Candida glabrata. Ckru is Candida krusei. The shaded boxes thatspan all 8 sequences indicate the regions that were used in the Examplesto design primers that amplify part of the 18S region of all eight ofthese Candida species. The primers corresponding to these regions areshown in Table 1 as SEQ ID NOs: 31 (forward) and (reverse) 32. Thenon-shaded boxes on the sequence for Candida krusei correspond todifferent regions that can be used to design primers that amplify partof the 18S region for this species. The primers corresponding to theseregions are shown in Table 1 as SEQ ID NOs: 41 (forward) and 42(reverse). The shaded boxes that only span the sequence of one speciesindicate regions that are specific for the particular species ofCandida. These regions (SEQ ID NOs: 30, 34, 36, 38 and 40) can be usedto design species-specific probes. The molecular beacon probes used todetect the different species-specific regions are shown in Table 1 asSEQ. ID NOs: 33, 35, 37, 39 and 43.

FIG. 6 shows the specific detection of Candida tropicalis DNA (seearrow) in a multiplex reaction.

FIG. 7 shows the specific detection of Candida parapsilosis DNA (seearrow) in a multiplex reaction.

FIG. 8 shows the specific detection of Candida albicans DNA (see arrow)in a multiplex reaction.

FIG. 9 shows the specific detection of Candida glabrata DNA (see arrow)in a multiplex reaction.

FIG. 10 shows the specific detection of Candida krusei DNA (see arrow)in a multiplex reaction.

FIG. 11 shows the structure of Cy®5 mono NHS ester.

FIG. 12 shows the structure of Texas Red®.

FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G, 13H, 13I, 13J, 13K, 13L, 13Mand 13N depict graphical displays of real-time PCR results frommultiplex panel experiments (see Example 7).

FIG. 14 shows the Limit of Detection for Aspergillus for a multiplexassay designed to detect Aspergillus genus, Pneumocystis jirovecii andan internal control.

FIG. 15 shows the Limit of Detection for Pneumocystis for a multiplexassay designed to detect Aspergillus genus, Pneumocystis jirovecii andan internal control.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the panfungal sequence that is detected in Examples 1to 4.

SEQ ID NOs: 2 and 3 show the panfungal oligonucleotide primers used inExamples 1 to 4.

SEQ ID NO: 4 shows the panfungal molecular beacon probe used in Examples1 to 4.

SEQ ID NO: 5 shows the Candida-specific sequence that is detected inExample 3.

SEQ ID NOs: 6 and 7 show the Candida oligonucleotide primers used inExample 3.

SEQ ID NO: 8 shows the Candida molecular beacon probe used in Example 3.

SEQ ID NO: 9 shows the sequence of the portion of the Maize (Zea mayis)tRNA-LEU intron region used as an internal control in the Examples.

SEQ ID NO: 10 shows the sequence within SEQ ID NO: 9 that is detected aspart of the internal control in the Examples.

SEQ ID NOs: 11 and 12 show the oligonucleotide primers used as part ofthe internal control in the Examples.

SEQ ID NO: 13 shows the molecular beacon probe used to detect SEQ ID NO10 as part of the internal control in the Examples.

SEQ ID NO: 14 shows the Aspergillus-specific sequence that is detectedin Examples 3 and 4.

SEQ ID NOs: 15 and 16 show the Aspergillus oligonucleotide primers usedin Examples 3 and 4.

SEQ ID NO: 17 shows the Aspergillus molecular beacon probe used inExamples 3 and 4.

SEQ ID NO: 18 shows the Pneumocystis-specific sequence that is detectedin Example 4.

SEQ ID NOs: 19 and 20 show the Pneumocystis oligonucleotide primers usedin Example 4.

SEQ ID NO: 21 shows the Pneumocystis molecular beacon probe used inExample 4.

SEQ ID NO: 22 shows the sequence of 18S in Candida tropicalis

SEQ ID NO: 23 shows the sequence of 18S in Candida parapsilosis.

SEQ ID NO: 24 shows the sequence of 18S in Candida dubliniensis.

SEQ ID NO: 25 shows the sequence of 18S in Candida albicans.

SEQ ID NO: 26 shows the sequence of 18S in Candida guilliermondii.

SEQ ID NO: 27 shows the sequence of 18S in Candida lusitaniae.

SEQ ID NO: 28 shows the sequence of 18S in Candida glabrata.

SEQ ID NO: 29 shows the sequence of 18S in Candida krusei.

SEQ ID NO: 30 shows the Candida tropicalis-specific sequence that isdetected in Example 6.

SEQ ID NO: 31 shows the sequence of the forward primer that is used inExample 6 to amplify part of the 18S region for all five Candidaspecies.

SEQ ID NO: 32 shows the sequence of the reverse primer that is used inExample 6 to amplify part of the 18S region for all five Candidaspecies.

SEQ ID NO: 33 shows the sequence of the molecular beacon probe that isused in Example 6 to specifically detect Candida tropicalis.

SEQ ID NO: 34 shows the Candida parapsilosis-specific sequence that isdetected in Example 6.

SEQ ID NO: 35 shows the sequence of the molecular beacon probe that isused in Example 6 to specifically detect Candida parapsilosis.

SEQ ID NO: 36 shows the Candida albicans-specific sequence that isdetected in Example 6.

SEQ ID NO: 37 shows the sequence of the molecular beacon probe that isused in Example 6 to specifically detect Candida albicans.

SEQ ID NO: 38 shows the Candida glabrata-specific sequence that isdetected in Example 6.

SEQ ID NO: 39 the sequence of the molecular beacon probe that is used inExample 6 to specifically detect Candida glabrata.

SEQ ID NO: 40 shows the Candida krusei-specific sequence that isdetected in Example 6.

SEQ ID NO: 41 shows the sequence of the forward primer that can be usedto amplify part of the 18S region for all five species of Candida.

SEQ ID NO: 42 shows the sequence of the reverse primer that can be usedto amplify part of the 18S region for all five species of Candida.

SEQ ID NO: 43 shows the sequence of the molecular beacon probe that isused in Example 6 to specifically detect Candida krusei.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedmethods may be tailored to the specific needs in the art. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “amethod” includes “methods”, reference to “a probe” includes two or moresuch probes, reference to “a label” includes two or more such labels,reference to “fungus” includes two or more fungi, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The invention generally concerns the rapid detection and identificationof a fungus or more than one fungus in a sample. The invention allowsthe rapid detection of any fungus in a sample (panfungal detection). Theinvention in a preferred embodiment allows the rapid detection andidentification of a fungus belonging to the genus Candida, Aspergillusor Pneumocystis. In still another preferred embodiment of the invention,a method is provided for the rapid detection and identification of afungus belonging to the species Candida tropicalis, Candidaparapsilosis, Candida albicans, Candida glabrata or Candida krusei.

A Description of Selected Embodiments of the Invention

In one aspect, a method of testing for the presence or absence of anyfungus in a sample is provided, comprising obtaining a sample suspectedof containing fungal nucleic acid, including at least one universalregion of fungal nucleic acid, and testing for the presence or absencein the sample of the at least one universal region of fungal nucleicacid. In some embodiments, the nucleic acid comprises DNA, and in otherembodiments, the nucleic acid comprises RNA.

Samples may be obtained from biological or non-biological sources. Forexample the biological source samples can include, but are not limitedto, a biological fluid, tissue, or a combination of any two or morethereof. Non-biological sources may include, but are not limited tosamples obtained from the environment. For example, some non-biologicalsources may include an air sample, a water sample, a soil sample, orcombinations thereof. Non-biological sources may also include a piece ofa vehicle, watercraft, aircraft, building, or dwelling.

In some embodied methods, the at least one universal region of fungalnucleic acid includes SEQ ID NO: 1, a complement or a transcriptthereof, or a sequence having 80% or more sequence homology with SEQ IDNO: 1, the complement or the transcript thereof. In other embodiments,the at least one universal region of fungal nucleic acid includes SEQ IDNO: 1, a complement or a transcript thereof, or a sequence having 90% ormore sequence homology with SEQ ID NO: 1, complement or transcriptthereof.

In some embodied methods the testing step includes contacting the samplewith an oligonucleotide probe comprising a nucleic acid capable ofhybridizing to the at least one universal region of fungal nucleic acidunder stringent conditions. The testing step may also include contactingthe sample with an oligonucleotide probe comprising a nucleic acidcapable of hybridizing to the at least one universal region of fungalnucleic acid under non-stringent conditions. In such methods, theoligonucleotide probe may further include a detectable label. In othersuch methods, the probe includes SEQ ID NO: 4, complement or transcriptthereof, or a sequence having 90% or more sequence homology with SEQ IDNO: 4, complement or transcript thereof

Methods embodied herein may further comprise amplifying the at least oneuniversal region of fungal nucleic acid. For example, the amplifyingstep may include contacting the sample with a pair of primers such as,but not limited to, SEQ ID NO: 2 and SEQ ID NO: 3.

In methods in which the universal region of the fungal nucleic acid isamplified, the amplifying step may be carried out in the presence of oneor more internal PCR amplification controls to ensure appropriateamplification of any fungal nucleic acid present in the sample. In suchmethods the one or more internal PCR amplification controls may comprisea non-fungal sequence. In other such methods, the amplifying step may becarried out in the presence of a cloned or synthesized tRNA-LEU intronregion, which is added to the amplification mixture in a predeterminedamount to rule out the presence of inhibitors or other defectiveamplification steps. tRNA-LEU intron regions used in the embodiedmethods may comprise a portion of the Maize (Zea mayis) tRNA-LEU intronregion, for example, including but not limited to, SEQ ID NO: 9. In somesuch embodied methods, the method further comprises detecting thepresence of a nucleic acid including SEQ ID NO: 10. In other suchembodied methods, the detecting step comprises contacting the samplewith a pair of oligonucleotides primers including SEQ ID NO: 11 and SEQID NO: 12 and a molecular beacon probe including SEQ ID NO: 13.

In another aspect, a method is provided for testing for the presence orabsence of a fungus belonging to the genus Candida in a sample. Suchmethods comprise obtaining a sample suspected of containing fungalnucleic acid, including at least one region of fungal nucleic acidcharacteristic of the genus Candida, and testing for the presence orabsence in the sample of the at least one region of fungal nucleic acidcharacteristic of the genus Candida. In some embodiments, the at leastone region of fungal nucleic acid characteristic of the genus Candidaincludes SEQ ID NO: 5, a complement or transcript thereof, or a sequencehaving 80% or more sequence homology with SEQ ID NO: 5, complement ortranscript thereof.

In other embodiments, the testing step includes contacting the samplewith a probe including SEQ ID NO: 8. In some such embodiments themethods further comprise amplifying the at least one region of fungalnucleic acid characteristic of the genus Candida by contacting thesample with a pair of primers including SEQ ID NO: 6 and SEQ ID NO: 7.

In another aspect, a method of testing for the presence or absence of afungus belonging to the genus Aspergillus in a sample is provided,comprising obtaining a sample suspected of containing fungal nucleicacid, including at least one region of fungal nucleic acidcharacteristic of the genus Aspergillus, and testing for the presence orabsence in the sample of the at least one region of fungal nucleic acidcharacteristic of the genus Aspergillus.

In some embodiments, the at least one region of fungal nucleic acidcharacteristic of the genus Aspergillus includes SEQ ID NO: 14, acomplement or transcript thereof, or a sequence having 80% or moresequence homology with SEQ ID NO: 14, complement or transcript thereof.In some such methods, the testing step includes contacting the samplewith a probe including SEQ ID NO: 17. Such methods may further compriseamplifying the at least one region of fungal nucleic acid characteristicof the genus Aspergillus by contacting the sample with a pair of primersincluding SEQ ID NO: 15 and SEQ ID NO: 16.

In yet another aspect, a method of testing for the presence or absenceof a fungus belonging to the genus Pneumocystis in a sample is provided,comprising obtaining a sample suspected of containing fungal nucleicacid, including at least one region of fungal nucleic acidcharacteristic of the genus Pneumocystis, and testing for the presenceor absence in the sample of the at least one region of fungal nucleicacid characteristic of the genus Pneumocystis.

In some embodied methods, the at least one region of fungal nucleic acidcharacteristic of the genus Pneumocystis includes SEQ ID NO: 18, acomplement or transcript thereof, or a sequence having 80% or moresequence homology with SEQ ID NO: 18, complement or transcript thereof.In some such methods, the testing step includes contacting the samplewith a probe including SEQ ID NO: 21. Some such methods may furthercomprise amplifying the at least one region of fungal nucleic acidcharacteristic of the genus Pneumocystis by contacting the sample with apair of primers including SEQ ID NO: 19 and SEQ ID NO: 20.

In yet another aspect, a method of testing for the presence or absenceof a fungus belonging to the species Candida tropicalis in a sample isprovided, comprising obtaining a sample suspected of containing fungalnucleic acid, including at least one region of fungal nucleic acidcharacteristic of the species Candida tropicalis, and testing for thepresence or absence in the sample of the at least one region of fungalnucleic acid characteristic of the species Candida tropicalis. In someembodied methods, the at least one region of fungal nucleic acidcharacteristic of the species Candida tropicalis includes SEQ ID NO: 30,a complement or transcript thereof, or a sequence having 80% or moresequence homology with SEQ ID NO: 30, complement or transcript thereof.The testing step may include contacting the sample with a probeincluding SEQ ID NO: 33.

In yet another aspect, a method of testing for the presence or absenceof a fungus belonging to the species Candida parapsilosis in a sample isprovided, comprising obtaining a sample suspected of containing fungalnucleic acid, including at least one region of fungal nucleic acidcharacteristic of the species Candida parapsilosis, and testing for thepresence or absence in the sample of the at least one region of fungalnucleic acid characteristic of the species Candida parapsilosis. In someembodied methods, the at least one region of fungal nucleic acidcharacteristic of the species Candida parapsilosis includes SEQ ID NO:34, a complement or transcript thereof, or a sequence having 80% or moresequence homology with SEQ ID NO: 34, complement or transcript thereof.The some such methods, the testing step may include contacting thesample with a probe including SEQ ID NO: 35.

In yet another aspect, a method of testing for the presence or absenceof a fungus belonging to the species Candida albicans in a sample,comprising obtaining a sample suspected of containing fungal nucleicacid, including at least one region of fungal nucleic acidcharacteristic of the species Candida albicans, and testing for thepresence or absence in the sample of the at least one region of fungalnucleic acid characteristic of the species Candida albicans. In someembodied methods, the at least one region of fungal nucleic acidcharacteristic of the species Candida albicans includes SEQ ID NO: 36, acomplement or transcript thereof, or a sequence having 80% or moresequence homology with SEQ ID NO: 36, complement or transcript thereof.In some such methods the testing step may include contacting the samplewith a probe including SEQ ID NO: 37.

In another aspect, a method of testing for the presence or absence of afungus belonging to the species Candida glabrata in a sample isprovided, comprising obtaining a sample suspected of containing fungalnucleic acid, including at least one region of fungal nucleic acidcharacteristic of the species Candida glabrata, and testing for thepresence or absence in the sample of the at least one region of fungalnucleic acid characteristic of the species Candida glabrata. In someembodied methods the at least one region of fungal nucleic acidcharacteristic of the species Candida glabrata includes SEQ ID NO: 38, acomplement or transcript thereof, or a sequence having 80% or moresequence homology with SEQ ID NO: 38, complement or transcript thereof.In some such methods the testing step may include contacting the samplewith a probe including SEQ ID NO: 39.

In yet another aspect, a method of testing for the presence or absenceof a fungus belonging to the species Candida krusei in a sample isprovided, comprising obtaining a sample suspected of containing fungalnucleic acid, including at least one region of fungal nucleic acidcharacteristic of the species Candida krusei, and testing for thepresence or absence in the sample of the at least one region of fungalnucleic acid characteristic of the species Candida krusei. In someembodied methods, the at least one region of fungal nucleic acidcharacteristic of the species Candida krusei includes SEQ ID NO: 40, acomplement or transcript thereof, or a sequence having 80% or moresequence homology with SEQ ID NO: 40, complement or transcript thereof.In such methods the testing step may include contacting the sample witha probe including SEQ ID NO: 43.

In the above embodied methods in which the fungus belongs to a Candidaspecies, the methods may further comprise amplifying the at least oneregion of fungal nucleic acid characteristic of the Candida species bycontacting the sample with a pair of primers including SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 41, and SEQ ID NO: 42.

In other aspects, the inventive methods can be employed in multiplexreactions to simultaneously test for the presence or absence of avariety of fungi. One or more of the panfungal detection methodsdescribed below can be carried out simultaneously in a multiplexreaction with one or more of the genus detection methods described belowand/or one of the species detection methods described below. Similarly,one or more of the genus detection methods described below can becarried out simultaneously in a multiplex reaction with one or more ofthe species detection methods described below. For example, theinventive methods can be used to simultaneously evaluate a sample forthe presence of fungi generally, Pneumocystis, Aspergillus, as well asan internal control. In another example, the inventive methods can beused to simultaneously detect in a sample Aspergillus genus, Pnemocystisjirovecii and an internal control.

Testing kits are also provided for testing for a number of fungalnucleic acids. In some embodiments, a panfungal nucleic acid testing kitis provided comprising a molecular beacon probe including SEQ ID NO: 4and a pair of primers including SEQ ID NO: 2 and SEQ ID NO: 3. In otherembodiments, a kit for testing for the presence or absence of at leastone region of fungal nucleic acid characteristic of the genus Candida ina sample is provided, comprising a molecular beacon probe including SEQID NO: 8 and a pair of primers including SEQ ID NO: 6 and SEQ ID NO: 7.

In yet other embodiments, a kit for testing for the presence or absenceof at least one region of fungal nucleic acid characteristic of thegenus Aspergillus in a sample is provided, comprising a molecular beaconprobe including SEQ ID NO: 17 and a pair of primers including SEQ ID NO:15 and SEQ ID NO: 16.

In yet other embodiments, a kit for testing for the presence or absenceof at least one region of fungal nucleic acid characteristic of thegenus Pneumocystis in a sample is provided, comprising a molecularbeacon probe including SEQ ID NO: 21 and a pair of primers including SEQID NO: 19 and SEQ ID NO: 20.

In yet other embodiments, a kit for testing for the presence or absenceof at least one region of fungal nucleic acid characteristic of aspecies of Candida in a sample is provided, comprising one or moremolecular beacon probes including SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 43, or combinations thereof and a pairof primers including SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 41 andSEQ ID NO: 42, or both pairs. Such kits may further comprise one or moreinternal PCT amplification controls.

Panfungal Detection Method

The invention provides a method for the rapid detection of the presenceor absence of any fungus in a sample. This is referred to herein as thepanfungal detection method. The panfungal detection method involvesdetermining whether or not a sample comprises any

fungus. The method therefore involves determining whether or not asample comprises a fungus that belongs to any fungal phylum, any fungalgenus or any fungal species. The method can be used in relation to anyfungus. The fungus can belong to any of the five fungal phyla,Chytridiomycota, Zygomycota, Glomeromycota, Ascomycota or Basidiomycota.The fungus can be mould, yeast, smut or mushroom. The fungus ispreferably a fungus that is capable of infecting humans or animals. Thefungus can belong to any fungal genus. The fungus preferably belongs tothe genus Candida, Aspergillus, Pneumocystis, Cryptococcus, Blastomyces,Coccidioides, Paracoccidioides, Penicillium, Mucor, Scedosporium,Saccharomyces, Histoplasma, Fusarium, Paecilomyces, Trichosporon,Acremonium, Rhizopus, Rhizomucor, Mucor, Cuninghamella, Malezzesia,Blastoschizomyces, Scedosporium, Goetrichum, Trichophyton, Exophiala,Exserohilum, Fonsecea, Cladosporium, Curvularia, BasidiobolusAureobasidium, Schizophyllum, Sporothrix, Scopulariopsis or Absidia. Thefungus can belong to any fungal species. The fungus can belong to any ofthe species exemplified below.

The panfungal detection method gives an indication of thefungus-containing status of the sample. The method indicates either thatthere is a fungus or fungi present in the sample or that there is nofungus present in the sample. The method allows the detection of afungus in a sample without the need to test for a specific fungal genusor for a specific fungus species.

The panfungal detection method can be carried out on any sample. In oneembodiment, the sample is a non-biological sample. Specific types ofsample are discussed in more detail below. The panfungal detectionmethod is typically carried out on a sample whose fungus-containingstatus is not known. In other words, the panfungal detection method istypically carried out when it is not known whether or not a samplecontains a fungus. The panfungal detection method is preferably carriedout on a sample that is suspected of containing a fungus. The method canbe carried out on a sample that is known to contain a fungus to confirmthe presence of the fungus.

The panfungal detection method comprises detecting the presence orabsence in the sample of at least one universal region of fungal DNA.The presence of a universal region in the sample is indicative of thepresence of a fungus in the sample. The absence of a universal regionfrom the sample is indicative of the absence of a fungus from thesample. The method may involve detecting 1, 2, 3, 4, 5, 10, 15, 20 ormore universal regions. A universal region of fungal DNA (hereinafter“universal region”) is a region of DNA that is present in the genomes ofall fungi. The universal region is not present in any other organism,particularly a micro-organism such as a bacteria or a virus. Forexample, a universal region is region of DNA that is present in allfungi but that is not present in Escherichia coli and Staphylococcusaureus. A region of fungal DNA is a DNA sequence within the genome of afungus. The region can be part of a gene, such as an intron, an exon ora part thereof. Alternatively, the region can incorporate a part of oneor more genes.

The universal region is typically at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 15, at least 20, at least 25,at least 30, at least 35, at least 45, at least 50, at least 75 or atleast 100 nucleotides in length. For example, the universal region canbe from 5 to 200, from 7 to 100 or from 10 to 50 nucleotides in length.The region is preferably 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides inlength.

In one embodiment, only the universal region is detected. In anotherembodiment, the universal sequence is detected as part of largersequence. For instance, the universal region can be detected as part ofa sequence that has flanking sequences at the 5′ end, the 3′ end or atboth the 5′ and 3′ ends of the universal region. The flanking sequencescan be at least 1, at least 2, at least 5, at least 10, at least 15, atleast 20, at least 25, at least 30, at least 35, at least 45, at least50, at least 75 or at least 100 nucleotides in length. For example, theflanking regions can be from 1 to 100, from 5 to 50 or from 10 to 20nucleotides in length.

The universal region is preferably part of the 18S region.

The universal region is preferably a sequence that shares at least 80%,at least 85%, at least 90%, at least 95%, at least 98% or at least 99%homology or sequence identity with SEQ ID NO: 1. The universal region iseven more preferably a sequence that is identical to SEQ ID NO: 1.

In another embodiment, the panfungal detection methods involve detectingthe presence or absence in a sample of at least one universal region offungal DNA present in at least the following fungi: Gymnopus spp.,Rhodocollybia butyracea, Hypholoma fasciculare, Saccharomycescerevisiae, Tuber spp., Bothia castanella, Rhizosphere spp.,Herpotrichiellaceae spp., Verrucariaceae spp., Marchandiomyces spp.,Minimedusa spp., Marchandiobasidium aurantiacum, Marchandiomycescorallinus, Marchandiomyces lignicola, Burgoa spp., Athelia arachnoidea,Alternaria alternata, Alternaria spp., Boletus edulis, Leccinumaurantiacum, Trametes versicolor, Trametes spp., Sympodiomycopsis spp.,Flavocetraria nivalis, Ampelomyces spp., Gymnopus biformis, Gymnopusspp., Gymnopus confluens, Gymnopus spongiosus, Collybia readii,Marasmiellus stenophyllus, Marasmiellus ramealis, Marasmius scorodonius,Collybia marasmioides, Micromphale brassicolens, Caripia montagnei,Rhodocollybia spp., Anthracophyllum lateritium, Anthracophyllum archeri,Anthracophyllum spp., Phanerochaete spp., Schizosaccharomyces pombe,Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus,Aspergillus niger, Aspergillus spp., Tricholoma imbricatum, Tricholomaflavovirens, Tomentella sublilacina, Rhizopogon spp., Laccaria spp.,Inocybe spp., Hebeloma spp., Cortinarius spp., Clavulina spp., Xerocomusspp., Amanita spp., Eurotium herbariorum, Edyuillia athecia, Warcupiellaspinulosa, Hemicarpenteles paradoxus, Hemicarpenteles acanthosporus,Hemicarpenteles spp., Chaetosartorya cremea, Petromyces spp., Graphiumtectonae, Di plolaimelloides spp., Rhabdolaimus spp., Hohenbueheliapetalodes, Glomerella graminicola, Cryptococcus arboriformis,Cryptococcus neoformans, Cryptococcus spp., Gamsylella parvicollis,Monacrosporium haptotylum, Monacrosporium sichuanense, MonacrosporiumSpp., Monacrosporium gephyropagum, Monacrosporium spp., Drechslerellacoelobrocha, Drechslerella dactyloides, Drechslerella spp., Arthrobotrysmusiformis, Arthrobotrys flagrans, Arthrobotrys hertziana, Arthrobotrysoligospora, Arthrobotrys vermicola, Arthrobotrys spp., Monacrosporiumdrechsleri, Vermispora spp., Pseudallescheria boydii (Scedosporiumapiospermum), Scedosporium inflatum, Geosmithia spp., Glomerellacingulata, Lophodermium piceae, Fusarium asiaticum, Fusarium spp.,Pleurotus eryngii, Cintractia sorghi-vulgaris, Cantharocybe gruberi,Bourdotia spp., Auricularia spp., Puccinia bartholomaei, Puccinia spp.,Diaporthe phaseolorum, Melanconis stilbostoma, Xylaria spp.,Trichophyton equinum, Trichophyton tonsurans, Trichophytum violaceum,Trichophytum rubrum, Trichophytum interdigitale, Trichophytumschoenleinii Trichophyton spp., Chlorophyllum agaricoides, Cenococcumgeophilum, Helotiales spp., Rhizoscyphus ericae, Lactarius pubescens,Lactarius spp., Piloderma fallax, Suillus luteus, Amanita muscaria,Tricholoma spp., Laccaria cf bicolour, Cortinarius purpurascens,Seiridium spp., Apiospora montagnei, Chondrostereum purpureum,Botryobasidium subcoronatum, Boletellus shichianus, Boletellus spp.,Hypocrea farinose, Hypocrea spp., Sarcostroma restionis, Sarcostromaspp., Truncatella betulae, Truncatella spp., Pestalotiopsis matildae,Paraconiothyrium spp., Phoma spp., Cunninghamella bainieri,Cunninghamella bertholletiae, Cantharellus cibarius, Apiospora bambusae,Apiospora spp., Discostroma botan, Cercophora caudate, Gnomoniaribicola, Faurelina elongate, Mycorrhiza fungi, Geomyces pannorum,Coprinus spp., Acremonium spp., Clonostachys spp., Phoma eupyrena,Tetracladium spp., Mortierella spp., Tulasnella calospora, Epulorhizaspp., Tulasnella calospora, Antarctomyces psychrotrophicus,Amphisphaeriaceae spp., Phomopsis spp., Trichoderma spp., Pestalotiopsisspp., Pestalotiopsis spp., Trichocomaceae spp., Coniochaetales spp.,Tremellales spp., Dothideales spp., Phyllachoraceae spp.,Saccharomycetales spp., Herpotrichiellaceae spp., Liliopsida spp.,Trichosporonales spp., Trichosporon mycotoxinivorans, Trichosporon spp.,Dothioraceae spp., Hypocreales spp., Mycosphaerellaceae spp.,Sporidiobolales spp., Clavicipitaceae spp., Pleosporales spp.,Ustilaginaceae spp., Phyllachoraceae spp., Mucoraceae spp., Sordarialesspp., Filobasidiales spp., Calosphaeriaceae spp., Clavicipitaceae spp.,Mucorales spp., Herpotrichiellaceae spp., Microdochium spp.,Phyllachoraceae spp., Zopfiaceae spp., Botryosphaeriaceae spp.,Helotiaceae spp., Bionectriaceae spp., Lachnocladiaceae spp., Dipodascaceae spp., Caulerpaceae spp., Microstromatales spp.,Aphyllophorales spp., Montagnulaceae spp., Gymnoascaceae spp.,Cryphonectriaceae spp., Xylariales spp., Montagnulaceae spp.,Chaetomiaceae spp., Xanthoria elegans, Rhizopus spp., Penicillium spp.,Cetraria aculeate, Nephromopsis laureri, Tuckermannopsis chlorophylla,Cetraria ericetorum, Cetraria spp., Flavocetraria cucullata,Kaernefeltia merrillii, Amorosia littoralis, Quambalaria cyanescens,Cordyceps roseostromata, Cordyceps spp., Russula spp., Clavulina spp.,Tuber quercicola, Gymnomyces spp., Tetrachaetum elegans, Anguillosporalongissima, Hypocrea spp., Sirococcus conigenus, Rhizopogon roseolus,Rhizopogon olivaceotinctus, Rhizopogon spp., Pisolithus microcarpus,Rhizoscyphus ericae, Cortinarius glaucopus, Paxillus spp., Suillusvariegates, Pyrobaculum aerophilum, Tulasnella spp., Hohenbuehelia spp.,Cochliobolus lunatus, Plicaturopsis crispa, Bondarcevomyces taxi,Tapinella panuoides, Tapinella spp., Austropaxillus spp., Gomphidiusroseus, Gyrodon lividus, Phylloporus pelletieri, Chamonixia caespitose,Porphyrellus porphyrosporus, Truncocolumella citrina, Tapinellaatrotomentosa, Scleroderma leave, Suillus variegates, Suillus spp.,Porphyrellus porphyrosporus, Pisolithus arrhizus, Phaeogyroporusportentosus, Melanogaster variegates, Leucogyrophana mollusca,Hydnomerulius pinastri, Gomphidius roseus, Gyrodon lividus, Gyroporuscyanescens, Chalciporus piperatus, Chamonixia caespitose,Bondarcevomyces taxi, Dendryphiella triticicola, Guignardia spp.,Shiraia spp., Cladosporium spp., Phomopsis spp., Diaporthales spp.,Pestalotiopsis spp., Lophiostoma spp., Verticillium chlamydosporium,Paecilomyces lilacinus, Paecilomyces varioti, Paecilomyces spp.,Ceratorhiza oryzae-sativae, Geosmithia pallida, Geosmithia spp.,Geosiphon pyriformis, Agonimia spp., Pyrgillus javanicus, Exophialadermatitidis, Exophiala pisciphila, Exophiala spp., Ramichloridiumanceps, Ramichloridium spp., Capronia pilosella, Isaria farinose,Pochonia suchlasporia, Lecanicillium psalliotae, Dothideomycete spp.,Leotiomycete spp., Ustilaginoidea vixens, Hyphozyma lignicola,Coniochaeta malacotricha, Coniochaeta spp., Torrubiella confragosa,Isaria tenuipes, Microsporum canis, Microsporum audouinii, Microsporumspp., Epicoccum floccosum, Gigaspora rosea, Gigaspora spp., Ganodermaspp., Pseudoperonospora cubensis, Hyaloperonospora parasitica,Plectophomella spp., Aureobasidium pullulans, Gloeophyllum sepiarium,Gloeophyllum spp., Donkioporia expansa, Antrodia sinuosa,Phaeoacremonium rubrigenum, Phaeoacremonium spp., Albertiniellapolyporicola, Cephalotheca sulfurea, Fragosphaeria renifbrmis,Fragosphaeria spp., Phialemonium dimorphosporum, Phialemonium spp.,Pichia norvegensis, Pichia spp., Candida albicans, Candida tropicalis,Candida glabrata, Candida parapsilosis, Candida spp., Gondawanamycesspp., Graphium spp., Ambrosiella spp., Microglossum spp., Neobulgariapura, Holwaya mucida, Chlorovibrissea spp., Chlorociboria spp.,Thaxterogaster spp., Cortinarius spp., Setchelliogaster spp., Timgroveaspp., Descomyces spp., Hymenogaster arenarius, Quadrispora tubercularis,Quadrispora spp., Protoglossum violaceum, Ceratostomella pyrenaica,Ceratosphaeria lampadophora, Fonsecaea pedrosoi, Phlebia acerina,Phlebia spp., Pestalotiopsis disseminata, Paracoccidioides brasiliensis,Racospermyces koae, Endoraecium acaciae, Uromycladium tepperianum,Uromycladium spp., Agaricus bisporus, Agaricus spp., Psilocybequebecensis, Psilocybe merdaria, Psilocybe spp., Gymnopilus luteofolius,Gymnopilus liquiritiae, Gymnopilus spp., Hypholoma tuberosum, Melanotushartii, Panaeolus uliginosus, Stropharia rugosoannulata, Dermocybesemisanguinea, Dermocybe spp., Helicoma month* pes, Helicoma spp.,Tubeufia helicomyces, Tubeufia spp., Leohumicola verrucosa,Leptosphaerulina chartarum, Macrophoma spp., Marssonina rosae,Botryotinia fuckeliana, Pestalotiopsis spp., Chrysosporium carmichaelii,Chrysosporium spp., Dactylella oxyspora, Dactylellina lobatum,Cucurbitaceae spp., Chrysophyllum sparsiflorum, Chrysophyllum spp.,Blumeria graminis, Sawadaea polyfida, Sawadaea spp., Parauncinulaseptata, Erysiphe mori, Erysiphe spp., Typhulochaeta japonica,Golovinomyces orontii, Golovinomyces spp., Podosphaera xanthii,Podosphaera spp., Arthrocladiella mougeotii, Neoerysiphe galeopsidis,Phyllactinia kakicola, Phyllactinia spp., Cyphellophora laciniata,Sphaerographium tenuirostrum, Microsphaera trifolii, Sphaerothecaspiraeae, Sphaerotheca spp., Uncinuliella australiana, Absidiacorymbifera, Absidia spp., Geotrichum spp., Nectria curta, Anamikalactariolens, Hebeloma velutipes, Stropharia ambigua, Agrocybe praecox,Hydnum rufescens, Hydnum spp., Meliniomyces variabilis, Rhizoscyphusericae, Cryptosporiopsis ericae, Hyalodendron spp., Leptographiumlundbergii, Leptographium spp., Termitomyces spp., Coccidioidesposadasii, Coccidioides immitis, Sclerotinia sclerotiorum, Phomopsisspp., Metarhizium anisopliae, Cordyceps spp., Tilletiopsiswashingtonensis, Cerrena unicolor, Stachybotrys chartarum,Phaeococcomyces nigricans, Ganoderma philippii, Ganoderma spp.,Gloeophyllum sepiarium, Cystotheca lanestris, Leveillula taurica,Phyllactinia fraxini, Varicosporium elodeae, Rhinocladiella basitonum,Melanchlenus oligospermus, Clavispora lusitaniae, Rhizopus spp.,Phizomucor spp., Mucor spp., Conidiobolus coronatus, Conidobolus spp.,Basidiobolus ranarum, basidiobolus spp., Ochronis spp., Histoplasmacapsulatum, histoplasma spp., Wilcoxina mikolae, Lasiodiplodia spp.,Physcia caesia, Physcia spp., Brachyconidiellopsis spp., Conocybelacteal, Gastrocybe lateritia, Gastrocybe spp., Agrocybesemiorbicularis, Taphrina pruni, Taphrina spp., Asterophora parasitica,Asterophora spp., Eremothecium ashbyi, Tricladium splendens, Ramariaflava, Ramaria spp., Laccaria fraternal, Scutellospora spp., Illosporiumcarneum, Hobsonia christiansenii, Marchandiomyces corallinus, Fusicoccumluteum, Botryosphaeria ribis, Pseudozyma aphidis, Pseudozyma spp.,Pesotum erubescens, Battarrea stevenii, Battarrea spp., HarposporiumJanus, Harposporium spp., Hirsutella rhossiliensis, Arthrodermaciferrii, Arthroderma spp., Pucciniastrum goeppertianum, Cronartiumoccidentale, Cronartium arizonicum, Cronartium spp., Peridermiumharknessii, Peridermium spp., Chrysomyxa arctostaphyli, Holleyasinecauda, Holleya spp., Zoophthora radicans, Smittium culisetae,Auxarthron zuffianum, Renispora flavissima, Ctenomyces serratus, andSporothrix schenckii.

Standard methods in the art may be used to determine homology. Forexample, the UWGCG Package provides the BESTFIT program which can beused to calculate homology, for example used on its default settings(Devereux et al., Nucleic Acids Research, 1984; 12: 387395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul J Mol Evol, 1993; 36: 290-300; Altschul, et al (JMol Biol, 1990; 215: 403-10). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/)

In one embodiment, the universal region itself is detected. In anotherembodiment, RNA transcribed from the universal region is detected. Thepresence in the sample of RNA transcribed from a universal region isitself indicative of the presence of the universal region in the sample.The manner in which the universal region or any RNA transcribedtherefrom is detected is discussed in more detail below.

The universal region or the RNA transcribed therefrom is typicallyextracted from fungal cells present in the sample before it is detected.The universal region can be extracted using routine methods known in theart. For instance, suitable methods for extracting fungal DNA aredisclosed in Fredricks et al., J. Clin. Microbiol., 2005; 43(1):5122-5128, US Patent Application No. 2002/0115077, and U.S. Pat. No.6,605,439. Suitable methods of extracting fungal RNA are disclosed inthe art, such as the lithium chloride purification method disclosed inSambrook et al., 2001, Molecular Cloning: a laboratory manual, 3^(rd)edition, Cold Spring Harbour Laboratory Press. Kits for the extractionof fungal RNA, such as the RNeasy mini kit (Qiagen), are alsocommercially available.

The universal region can be detected using any method known in the art.The universal region is preferably detected using a probe thatspecifically hybridizes to the universal region. Typically, thedetecting comprises contacting the probe with the sample underconditions in which the probe specifically hybridizes to the region, ifpresent, and determining the presence or absence of the hybridizationproduct. The presence of the hybridization product indicates thepresence of the universal region. Conversely, the absence of thehybridization product indicates the absence of the universal region.

The probe is typically a nucleic acid, such as DNA, RNA, PNA or asynthetic nucleic acid. A probe specifically hybridizes to the universalregion if it preferentially or selectively hybridizes to the universalregion but does not hybridize to any other DNA or RNA sequences.

The probe preferably specifically hybridizes to the universal regionunder stringent conditions. Conditions that permit the hybridization arewell-known in the art (for example, Sambrook et al., 2001, MolecularCloning: a laboratory manual, 3″^(I) edition, Cold Spring HarbourLaboratory Press; and Current Protocols in Molecular Biology, Chapter 2,Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York(1995)). Detection can be carried out under low stringency conditions,for example in the presence of a buffered solution of 30 to 35%formamide, 1 M NaCl and 1% SDS (sodium dodecyl sulfate) at 37° C.followed by a wash in from 1× (0.1650 M Na^(t)) to 2× (0.33 M Na^(t))SSC (standard sodium citrate) at 50° C. Detection can be carried outunder moderate stringency conditions, for example in the presence of abuffer solution of 40 to 45% formamide, 1 M NaCl, and 1 SDS at 37° C.,followed by a wash in from 0.5× (0.0825 M Na^(t)) to 1× (0.1650 MNa^(t)) SSC at 55° C. Detection can be carried out under high stringencyconditions, for example in the presence of a buffered solution of 50%formamide, 1 M NaCl, 1% SDS at 37° C., followed by a wash in 0.1×(0.0165 M Na^(t)) SSC at 60° C.

The probe can be the same length as, shorter than or longer than theuniversal region. The probe is typically at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20,at least 25, at least 30, at least 35, at least 45, at least 50, atleast 75 or at least 100 nucleotides in length. For example, the probecan be from 5 to 200, from 7 to 100, from 10 to 50 nucleotides inlength. The probe is preferably 5, 10, 15, 20, 25, 30, 35 or 40nucleotides in length. The probe preferably includes a sequence thatshares at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99% homology based on sequence identity with the universalregion. Homology can be determined as discussed above.

The probe is detectably-labelled. The detectable label allows thepresence or absence of the hybridization product formed by specifichybridization between the probe and the universal region (and therebythe presence or absence of the universal region) to be determined. Anylabel can be used. Suitable labels include, but are not limited to,fluorescent molecules, radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes,antibodies and linkers such as biotin.

The probe can be a scorpion probe, which is a probe linked to primer.The primer part of the probe can be designed to amplify the region offungal DNA to be detected and the probe part can designed to detect theamplified region. Scorpion probes are well-known in the art. They aredescribed in, for example, Whitcombe et al. (Nat. Biotechnol., 1994;117: 804-807).

The probe can be a molecular beacon probe. Molecular beacon probescomprise a fluorescent label at one end and a quenching molecule at theother. In the absence of the region to be detected, the probe forms ahairpin loop and the quenching molecule is brought into close proximitywith the fluorescent label so that no signal can be detected. Uponhybridization of the probe to the region to be detected, the loop unzipsand the fluorescent molecule is separated from the quencher such that asignal can be detected. Suitable fluorescent molecule and quenchercombinations for use in molecular beacons are known in the art. Suchcombinations include, but are not limited to, carboxyfluorsecein (FAM)and dabcyl. The probe is preferably the molecular beacon shown in SEQ IDNO: 4, SEQ ID NO: 44 or SEQ ID NO: 45.

The region of fungal DNA can be detected using TaqMan PCR. Thistechnique is well-known in the art.

The probe may be immobilised on a support using any technology which isknown in the art. Suitable solid supports are well-known in the art andinclude plates, such as multi well plates, filters, membranes, beads,chips, pins, dipsticks and porous carriers. The nanoparticles may beimmobilised on a support using any technology which is known in the art.

The detecting of the universal region preferably comprises the step ofamplifying the universal region or the RNA transcribed therefrom. In oneembodiment, the region is amplified before its presence is determined.In another embodiment, the region is detected in real time as itspresence is determined. Real-time methods are disclosed in the Examplesand have been described in the art. Such methods are described in, forexample, U.S. Pat. No. 5,487,972 and Afonia et al. (Biotechniques, 2002;32:946-9).

In one embodiment, only the region to be detected is amplified. In otherembodiments, the region to be detected is amplified as part of a muchlarger length of fungal DNA or RNA. Sequences of DNA or RNA having atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 150,at least 200, at least 250, at least 300, at least 400 or at least 500nucleotides and comprising the region to be detected can be amplified.For example, sequences having from 10 to 2000, from 20 to 1500, from 50to 1000 or from 100 to 500 nucleotides can be amplified.

The DNA or RNA can be amplified using routine methods that are known inthe art. The amplification of fungal DNA is preferably carried out usingpolymerase chain reaction (PCR) or nucleic acid sequence based analysis(NASBA). Suitable methods for PCR are disclosed in, for example, U.S.Pat. No. 6,605,439, EP-B-0979312 and Buchheidt et al. (British Journalof Haematology, 2002; 116:8030811).

A suitable method for the amplification of fungal DNA by NASBA isdescribed in Widjojoatmodjo et al., J. Microbiol. Methods, 1999;38(1-2):81-90.

Fungal RNA can be amplified using routine methods in the art, such asreverse transcription-PCR.

A person skilled in the art will be able to design specific primers toamplify the universal region. Primers are normally designed to becomplementary to sequences at either end of the sequence to be amplifiedbut not complementary to any other sequences. Primer design is discussedin, for example, Sambrook et al., 2001, supra.

Primers that amplify the 18S region are disclosed in, for example,Buchheidt et al. (British Journal of Haematology, 2002; 116: 8030811)and Makimura et al. (Japan J. Med. Sci. Biol., 1994; 47: 144-156.

The universal region is preferably amplified using (a) the primers shownin SEQ ID NOs: 2 and 3; or (b) one of the sense primers shown in SEQ IDNOs: 48 and 50 and one of the antisense primers shown in SEQ ID NOs: 49and 51.

The universal region or RNA transcribed therefrom is extracted fromfungal cells and so may be contaminated with one or factors thatinterfere with the amplification and/or detection steps. For thisreason, the universal region is typically detected in the presence of aninternal PCR amplification control. This ensures that any DNA present inthe sample is amplified correctly. The internal PCR amplificationcontrol preferably comprises a non-fungal sequence. The universal regionis preferably detected in the presence of a cloned or synthesizedtRNA-LEU intron region added to the amplification mixture in apredetermined amount to rule out the presence of inhibitors or otherdefective amplification steps. The tRNA-LEU intron region preferablycomprises a portion of the Maize (Zea mayis) tRNA-LEU intron region asshown in SEQ ID NO: 9. The sequence of the Maize (Zea mayis) tRNA-LEUintron region lacks homology with any sequence present in humans orpathogenic fungal species. The internal PCR amplification controlpreferably involves detecting a specific portion of SEQ ID NO: 9 asshown in SEQ ID NO: 10. SEQ ID NO: 10 can be detected using adetectably-labelled probe that specifically hybridizes to SEQ ID NO: 10as described above. The probe preferably includes a sequence that sharesat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99% homology based on sequence identity with SEQ ID NO: 10. Theprobe more preferably comprises a sequence that is identical to SEQ IDNO: 10. The detection of SEQ ID NO: 10 is most preferably carried outusing the pair of oligonucleotides primers shown in SEQ ID NOs: 11 and12 and the molecular beacon as shown in SEQ ID NO: 13. The primers arecapable of amplifying a sequence within the Maize (Zea mayis) tRNA-LEUintron region that contains SEQ ID NO: 10 as shown in Table 1 and themolecular beacon is capable of specifically detecting the presence ofSEQ ID NO: 10.

Genera Detection Methods

The invention also provides methods for the rapid detection of thepresence or absence in a sample of a fungus belonging to the genusCandida, Aspergillus or Pneumocystis. The methods give a rapidindication of whether or not a fungus belonging to the genus Candida,Aspergillus or Pneumocystis is contained within a sample. The methodsinvolve detecting in the sample a region of fungal DNA that is specificfor Candida, Aspergillus or Pneumocystis. The presence in a sample of aregion of fungal DNA that is specific for a particular genus isindicative of the presence of a fungus belonging to that genus in thesample. The absence from the sample of a region of fungal DNA that isspecific for a particular genus is indicative of the absence of a fungusbelonging to that genus from the sample. A region of DNA that isspecific for a genus is a region of DNA that is only present in thatgenus of fungi. Hence, a region of fungal DNA that is specific for agenus is present in the genomes of all the species of the genus but isnot present in the genomes of species of other fungal genera. A regionof fungal DNA that is specific for a genus is not present in the genomeof any other organism, particularly microorganisms such as a bacteria ora virus.

The methods for detecting the presence or absence of a fungus belongingto the genera Candida and Aspergillus can be carried out on any sample.The method for detecting the presence or absence of a fungus belongingto the genus Pneumocystis is carried out on a sample obtained from ahuman. Specific types of sample are discussed in more detail below. Inone embodiment, the methods are carried out on a sample that is known tocontain a fungus. For instance, the methods can be carried out on asample that has already undergone the panfungal detection methoddescribed above and a positive result was achieved. The methods can becarried out on a sample to confirm the identity of one or more fungiwhose presence in the sample is known. In another embodiment, themethods are carried on a sample whose fungus-containing status is notknown. The methods are typically carried out on a sample that issuspected of a fungus belonging to the genus Candida, Aspergillus orPneumocystis.

SEQ ID NO: 5 shows a region of fungal DNA that is specific for Candida.The method for detecting the presence or absence of a fungus belongingto the genus Candida comprises detecting in the sample the presence orabsence of a region of fungal DNA that shares at least 80% homology orsequence identity with SEQ ID NO: 5. The region preferably shares atleast 85%, at least 90%, at least 95%, at least 98% or at least 99%homology or sequence identity with SEQ ID NO: 5. The sequence is morepreferably identical to SEQ ID NO: 5. Homology can be determined asdescribed above.

This method can be used to detect the presence or absence of any speciesof fungus belonging to the genus Candida. The fungus can be Candidaalbicans, Candida dubliniensis, Candida famata, Candida glabrata,Candida guilliermondii, Candida haemulonii, Candida keyfyr, Candidakrusei, Candida lusitaniae, Candida hpolytica, Candida norvegensis,Candida parapsilosis, Candida tropicalis, Candida viswanathii, Candidainconspicua, Candida catenulata (also known as Candida brumptii andCandida ravautii), Candida pseudotropicalis, Candida parapsilosis,Candida metapsilosis, Candida orthopsilosis, Candida ciferrii, Candidafamata, Candida hpolytica, Candida norvegensis, Candida rugosa, Candidaviswanathii or Candida zeylanoides.

SEQ ID NO: 14 shows a region of fungal DNA that is specific forAspergillus. The method for detecting the presence or absence of afungus belonging to the genus Aspergillus comprises detecting in thesample the presence or absence of a region of fungal DNA that shares atleast 80% homology or sequence identity with SEQ ID NO: 14. The regionpreferably shares at least 85%, at least 90%, at least 95%, at least 98%or at least 99% homology or sequence identity with SEQ ID NO: 14. Thesequence is more preferably identical to SEQ ID NO: 14. Homology can bedetermined as described above.

This method can be used to detect the presence or absence of any speciesof fungus belonging to the genus Aspergillus. The fungus can beAspergillus alliaceus, Aspergillus alutaceus, Aspergillus atroviolaceus,Aspergillus caesiellus, Aspergillus candidus, Aspergillus carneus,Aspergillus chevalieri, Aspergillus clavato-nanicus, Aspergillusclavatus, Aspergillus conicus, Aspergillus deflectus, Aspergillusfischerianus, Aspergillus flavipes, Aspergillus flavus, Aspergillusfumigatus, Aspergillus glaucus, Aspergillus hollandicus, AspergillusJanus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus niger var. awamorii, Aspergillus niveus, Aspergillusochraceus, Aspergillus oryzae, Aspergillus penicilloides, Aspergillusreptans, Aspergillus restrictus, Aspergillus rubrobrunneus, Aspergillusspinosus, Aspergillus sydowii, Aspergillus tamarii, Aspergillus terreus,Aspergillus tetrazonus, Aspergillus unguis, Aspergillus ustus orAspergillus versicolor.

SEQ ID NO: 18 shows a region of fungal DNA that is specific forPneumocystis. The method for detecting the presence or absence of afungus belonging to the genus Pneumocystis comprises detecting in thesample the presence or absence of a region of fungal DNA that shares atleast 80% homology with SEQ ID NO: 18. The region preferably shares atleast 85%, at least 90%, at least 95%, at least 98% or at least 99%homology or sequence identity with SEQ ID NO: 18. The sequence is morepreferably identical to SEQ ID NO: 18. Homology can be determined asdescribed above.

This method can be used to detect the presence or absence of any speciesof fungus belonging to the genus Pneumocystis. The fungus can bePneumocystis carinii or Pneumocystis jirovecii.

In one embodiment, the region of fungal DNA that is specific forCandida, Aspergillus or Pneumocystis (i.e. that is homologous to SEQ IDNO: 5, 14 and 18) is itself detected. In another embodiment, any RNAtranscribed from the region is detected.

In one embodiment, only the region of fungal DNA is detected. In anotherembodiment, the region of fungal DNA is detected as part of largersequence. For instance, the region can be detected as part of a sequencethat has flanking sequences as described above with reference to thepanfungal detection method.

The region of fungal DNA or any RNA transcribed therefrom can beextracted as described above with reference to the panfungal detectionmethod.

The region of fungal DNA or any RNA transcribed therefrom can bedetected using any method described above with reference to thepanfungal method. The detecting of the region of fungal DNA preferablycomprises the step of amplifying the fungal DNA or any RNA transcribedtherefrom as described above with reference to the panfungal detectionmethod.

The region of fungal DNA that is specific for Candida (i.e. the regionthat is homologous to SEQ ID NO: 5) is preferably detected using themolecular beacon probe shown in SEQ ID NO: 8. The region of fungal DNAthat is specific for Candida is preferably amplified using the primersshown in SEQ ID NOs: 6 and 7.

The region of fungal DNA that is specific for Aspergillus (i.e. theregion that is homologus to SEQ ID NO: 14) is preferably detected usingthe molecular beacon probe shown in SEQ ID NO: 17. The region of fungalDNA that is specific for Aspergillus is preferably amplified using (a)the primers shown in SEQ ID NOs: 15 and 16; or (b) the primers shown inSEQ ID NOs: 46 and 47.

The region of fungal DNA that is specific for Pneumocystis (i.e. theregion that is homologus to SEQ ID NO: 18) is preferably detected usingthe molecular beacon probe shown in SEQ ID NO: 21. The region of fungalDNA that is specific for Pneumocystis is preferably amplified using theprimers shown in SEQ ID NOs: 19 and 20.

The molecular beacon probes used in any of the above genus detectionmethods are detectably-labelled. The molecular beacon probes can belabelled in any manner described above with reference to the panfungaldetection method.

The region of fungal DNA is typically detected in these methods in thepresence of an internal PCR amplification control as described abovewith reference to the panfungal detection method.

Two or three of the above methods can be carried out simultaneously.Hence, the presence or absence of fungi belonging to (i) Candida andAspergillus, (ii) Candida and Pneumocystis or (iii) Aspergillus andPneumocystis can be simultaneously detected in a sample. Similarly, thepresence or absence of fungi belonging to Candida, Aspergillus andPneumocystis can be simultaneously detected in a sample. Simultaneousdetection means that the fungi belonging to the two or more genera aredetected at the same time. The two or more genera are typically detectedin the same volume of sample. In other words, the two or more genera arenot typically detected in different aliquots of the sample. One, two orthree of the above methods for detecting the presence or absence offungi belonging to Candida, Aspergillus and Pneumocystis can be carriedout simultaneously in a multiplex reaction with one or more of thepanfungal detection methods described above and/or with one or more ofthe species detection methods described below. For instance, thepresence or absence of (i) any fungus (i.e. panfungal) and a fungusbelonging to Candida, (ii) any fungus and a fungus belonging toAspergillus, (iii) any fungus and a fungus belonging to Pneumocystis,(iv) any fungus, a fungus belonging to Candida and a fungus belonging toAspergillus, (v) any fungus, a fungus belonging to Candida and a fungusbelonging to Pneumocystis, or (vi) any fungus, a fungus belonging toAspergillus and a fungus belonging to Pneumocystis, can besimultaneously detected in a sample. Any of embodiments (i) to (vi) canfurther involve the simultaneous detection of the presence or absence ofone or more, such as 2, 3, 4 or 5, of Candida tropicalis, Candidaparapsilosis, Candida albicans, Candida glabrata or Candida krusei.Alternatively, the presence or absence of a fungus belonging to one, twoor three of Candida, Aspergillus and Pneumocystis and one or more, suchas 2, 3, 4 or 5, of Candida tropicalis, Candida parapsilosis, Candidaalbicans, Candida glabrata and Candida krusei can be simultaneouslydetected in a sample. The methods are typically carried outsimultaneously in the same volume of sample, i.e. they are not typicallycarried out in different aliquots of the sample.

If more than one of the methods are carried out simultaneously, thedifferent probes used to detect the different regions of fungal DNA(which indicate the presence or absence of a fungus or the differentgenera or species) are typically labelled with different labels. Probeshaving different labels are preferable when the different regions offungal DNA are being detected simultaneously in the same volume ofsample. When the two or more fungi are being detected in the same volumeof sample, it must be possible to distinguish between the differentlabels and hence detect the different regions of fungal DNA. Forinstance, fluorescent molecules that emit different wavelengths of lightcan be used. A suitable group of fluorescent labels, each of which canbe simultaneously detected, is HEX hexachlorofluorescein phosphoramidite(HEX), carboxyfluorescein (FAM), Cy®5 and Texas Red®. Other suitablegroups of labels are known in the art.

Species Detection Methods

The invention also provides methods for the rapid detection of thepresence or absence of in a sample of a fungus belonging to the speciesCandida tropicalis, Candida parapsilosis, Candida albicans, Candidaglabrata or Candida krusei. The fungus belonging to the species Candidaparapsilosis can belong to group I (also known as Candida parapsilosis),group II (also known as Candida orthopsilosis) or group III (also knownas Candida metapsilosis). The methods give a rapid indication of whetheror not a fungus belonging to the particular species are contained withina sample. The methods involve detecting in the sample a region of fungalDNA using specific molecular beacon probes. The specific molecularbeacon probes detect regions of fungal DNA that are specific for eachspecies. These regions are shown as shaded boxes that only span thesequence of one species in FIG. 5 (SEQ ID NOs: 30, 34, 36, 38 and 40). Aregion of DNA that is specific for a species is a region of DNA that isonly present in that species of fungi. Hence, a region of fungal DNAthat is specific for a species is not present in the genomes of otherspecies, even those of the same genus. A region of fungal DNA that isspecific for a genus is not present in the genome of any other organism,particularly micro-organisms such as a bacteria or a virus. The presencein the sample of the region of DNA that is specific for a particularspecies is indicative of the presence of a fungus belonging to thatspecies in the sample. The absence from the sample of the region of DNAthat is specific for a particular species is indicative of the absenceof a fungus belonging to that species from the sample.

The methods can be carried out on any sample. Specific types of sampleare discussed in more detail below. In one embodiment, the methods arecarried out on a sample that is known to contain a fungus as describedabove with reference to the panfungal detection method. In anotherembodiment, the methods are carried on a sample whose fungus-containingstatus is not known. The methods are typically carried out on a samplethat is suspected of a fungus belonging to the species Candidatropicalis, Candida parapsilosis, Candida albicans, Candida glabrata orCandida krusei.

In one embodiment, the region of fungal DNA is itself detected. Inanother embodiment, any RNA transcribed from the region is detected.

In one embodiment, only the region of fungal DNA is detected. In anotherembodiment, the region of fungal DNA is detected as part of largersequence. For instance, the region can be detected as part of a sequencethat has flanking sequences as discussed above with reference to thepanfungal detection method.

The region of fungal DNA or any RNA transcribed therefrom can beextracted as described above with reference to the panfungal detectionmethod.

The presence or absence of the region of fungal DNA that is specific forCandida tropicalis is detected using the molecular beacon probe shown inSEQ ID NO: 33. The presence or absence of the region of fungal DNA thatis specific for Candida parapsilosis is detected using the molecularbeacon probe shown in SEQ ID NO: 35. The presence or absence of theregion of fungal DNA that is specific for Candida albicans is detectedusing the molecular beacon probe shown in SEQ ID NO: 37. The presence orabsence of the region of fungal DNA that is specific for Candidaglabrata is detected using the molecular beacon probe shown in SEQ IDNO: 39. The presence or absence of the region of fungal DNA that isspecific for Candida krusei is detected using the molecular beacon probeshown in SEQ ID NO: 43. The molecular beacon probes aredetectably-labelled. The molecular beacon probes can be labelled in anymanner described above with reference to the panfungal detection method.

The detecting of the region of fungal DNA preferably comprises the stepof amplifying the fungal DNA or any RNA transcribed therefrom asdescribed above with reference to the panfungal detection method. Theregion of fungal DNA in any one of the five species is preferablyamplified using the primers shown in SEQ ID NOs: 31 and 32. The regionof fungal DNA in Candida krusei can be detected using the primers shownin SEQ ID NOs: 41 and 42.

The region of fungal DNA is typically detected in these methods in thepresence of an internal PCR amplification control as described abovewith reference to the panfungal detection method.

More than one of the above species detection methods can be carried outsimultaneously. In particular, 2, 3, 4 or 5 of the methods can becarried out simultaneously in any combination. Hence, the presence orabsence of fungi belonging to 2, 3, 4 or 5 of the particular species canbe simultaneously detecting in a sample. Simultaneous detection meansthat the fungi belonging to the two or more species are detected at thesame time. The two or more species are typically detected in the samevolume of sample. In other words, the two or more species are nottypically detected in different aliquots of the sample. One or more,such 2, 3, 4 or 5, of the above methods for detecting the presence orabsence of particular species of Candida can be carried outsimultaneously with one or more of the panfungal detection methodsdescribed above and/or one or more, such as 2 or 3, of the genusdetection methods described above. For instance, in addition to thecombinations described above, the presence or absence of any fungus(i.e. panfungal) and one or more, such as 2, 3, 4 or 5, of Candidatropicalis, Candida parapsilosis, Candida albicans, Candida glabrata andCandida krusei can be simultaneously detected in a sample. The methodsare typically carried out simultaneously in the same volume of sample,i.e. they are not typically carried out in different aliquots of thesample.

When more than one of the particular species is being detectedsimultaneously, the different regions of fungal DNA (which indicate thedifferent species) are preferably amplified using the primers shown inSEQ ID NOs: 31 and 32. These primers will amplify the appropriate regionin each of the five particular species of fungus.

If the two or three of the methods are carried out simultaneously, thedifferent probes used to detect the different regions of fungal DNA(which indicate the presence or absence of a fungus or the differentgenera or species) are typically labelled with different labels asdiscussed above with reference to the genus detection methods.

Sample

The sample used in the invention may be any suitable sample. Theinvention is typically carried out on a biological sample. The inventionis preferably carried out in vitro on a biological sample. Thebiological sample can be obtained from or extracted from any organism.The organism is typically eukaryotic and can belong the plantae kingdomor the animalia kingdom. The sample can be a colony of fungus.

The sample is preferably a fluid sample. The sample typically comprisesa body fluid. The sample may be urine, lymph, saliva, cerebrospinalfluid, peritoneal fluid, pericardial fluid, vitreous or other ocularsample, plural fluid, vaginal fluid, mucus, pus or amniotic fluid but ispreferably blood, plasma or serum. The sample can be a cell or tissuesample, such as lung, brain, liver, skin or nails.

Typically, the sample is human in origin, but alternatively it may benon-human. For instance, the sample can be from animals such as fromcommercially farmed animals such as horses, cattle, sheep or pigs or mayalternatively be pets such as cats or dogs. The sample can

also be from other organisms, such as insects. The sample can be from ahuman or non-human animal undergoing treatment with an anti-fungalagent.

The invention can also be carried out on a non-biological sample. Thenon-biological sample can be a fluid or a solid. Examples of anon-biological sample include surgical fluids, air, water such asdrinking water, reagents for laboratory tests and household containers.The sample may also be a particle collection device containing air,water, another liquid or a material.

The sample is typically processed prior to being used in the invention,for example by centrifugation or by passage through a membrane thatfilters out unwanted molecules or cells, such as red blood cells. Thesample may have undergone polymerase chain reaction before being used inthe invention. The sample may be measured immediately upon being taken.The sample may also be stored prior to assay, preferably below −70° C.

Kits

The invention also provides various kits for carrying out the methods ofthe invention. In particular, the invention provides a kit:

-   -   for the rapid detection of the presence or absence of any fungus        in a sample, comprising (a) the molecular beacon probe shown in        SEQ ID NO: 4 and the primers shown in SEQ ID NOs: 2 and 3; (b)        the molecular beacon probe shown in SEQ ID NO: 44, one or both        of the sense primers shown in SEQ ID NOs: 48 and 50 and one or        both of the antisense primers shown in SEQ ID NOs: 49 and 51;        or (c) the molecular beacon probe shown in SEQ ID NO: 45, one or        both of the sense primers shown in SEQ ID NOs: 48 and 50 and one        or both of the antisense primers shown in SEQ ID NOs: 49 and 51;    -   for the rapid detection of the presence or absence a fungus        belonging to the genus Candida in a sample, comprising the        molecular beacon probe shown in SEQ ID NO: 8 and the primers        shown in SEQ ID NOs: 6 and 7;    -   for the rapid detection of the presence or absence of a fungus        belonging to the genus Aspergillus in a sample, comprising the        molecular beacon probe shown in SEQ ID NO: 17 and (a) the        primers shown in SEQ ID NOs: 15 and 16 or (b) primers shown in        SEQ ID NOs: 46 and 47; and    -   for the rapid detection of the presence or absence of a fungus        belonging to the genus Pneumocystis in a sample, comprising the        molecular beacon probe shown in SEQ ID NO: 21 and the primers        shown in SEQ ID NOs: 19 and 20; and    -   for the rapid detection of the presence or absence of a fungus        belonging to a particular species of Candida in a sample,        comprising the primers shown in SEQ ID NOs: 31 and 32 or 41 and        42 and one or more of the molecular beacon probes shown in SEQ        ID NOs 33, 35, 37, 39 and 43.

The kit for the rapid detection of the presence or absence of a fungusbelonging to a particular species of Candida in a sample can comprise 1,2, 3, 4 or all 5 of the molecular beacon probes shown in SEQ ID NOs 33,35, 37, 39 and 43 in any combination.

The kits preferably further comprise reagents for extracting fungal DNAor RNA from a sample and/or primers that can be used to amplify theregion of fungal DNA and/or an internal control for the amplificationand detection stages. The internal control preferably comprises theportion of the Maize (Zea mayis) tRNA-LEU intron region shown in SEQ IDNO: 9, the pair of oligonucleotides primers shown in SEQ ID NOs: 11 and12 and the molecular beacon probe shown in SEQ ID NO: 13. The kit mayadditionally comprise one or more other reagents or instruments whichenable the method of the invention as described above to be carried out.Such reagents or instruments include one or more of the following:suitable buffer(s) (aqueous solutions), means to obtain a sample fromthe subject (such as a vessel or an instrument comprising a needle) or asupport comprising wells on which reactions can be done. Reagants may bepresent in the kit in a dry state such that a fluid sample resuspendsthe reagents. The kit may, optionally, comprise instructions to enablethe kit to be used in a method of the invention.

Uses of the Invention

The invention concerns the detection and identification of a fungus in asample. The invention can therefore be used for the diagnosis of afungal infection in a patient. The invention can also be used todetermine the presence of a fungus in or on any non-biological productand hence the likelihood that the product will cause a fungal infection.There are many situations in which it is important to ensure that anon-biological sample is fungus free. Examples include drinking waterand liquids used in laboratories. In particular, the invention may beused to pretest the components in a fungal diagnostic kit to ensure thecomponents are free from fungal nucleic acid (i.e. a quality controlstep). Other uses of the invention are clear to a person skilled in theart.

EXAMPLES

TABLE 1 the sequences of the primers and probes used in the Examples.I - Panfungal Sequence detected: TCGATTCCGGAGAGGGAGC (SEQ ID NO: 1)Sequence detected: CTGCGGCTTAATTTGACTCA (SEQ ID NO: 52)Sequence detected: ACATCCAAGGAAGGCAGCAG (SEQ ID NO: 53)Ia - Panfungal Oligonucleotide Primers 5′-GCCCTATCAACTTTCGATGG-3′(SEQ ID NO: 2) 5′-GCCTTCCTTGGATGTGGTAG-3′ (SEQ ID NO: 3)Ib Panfungal Molecular Beacon Probe5′-CGCGATTCGATTCCGGAGAGGGAGCATCGCG-3′ (SEQ ID NO: 4)(Hairpin structure nucleotides italicized) II CandidaSequence detected: GATGATTCATAATAACTTTTCG (SEQ ID NO: 5)IIa - Candida Oligonucleotide Primers 5′-TAGATAAAAAATCAATGCCTTCGG-3′(SEQ ID NO: 6) 5′-CATGGTAGGCCACTATCCTAC-3′ (SEQ ID NO: 7)IIb - Candida Molecular Beacon Probe5′-CGCGATGATGATTCATAATAACTTTTCGATCGCG-3′ (SEQ ID NO: 8)(Hairpin structure nucleotides italicized) III - Internal ControlPortion of the Maize (Zea mayis) tRNA-LEU intron region (SEQ ID NO: 9)  1 TAATGAATTCAATGATTCAAAAAAAACTAAGAGATGGATTAAATTATACAAGGAATCCTG 61 GTTTCAAAGAAAAGTAAAATGGGGATATGGCGAAATCGGTAGACGCTACGGACTTGATTG121 TATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGAGAAACCCTGG181 AATGAAAAATGGGCAATCCTGAGCCAAATCCCTTTTTTGAAAAACAAGTGGTTCTCAAAC241 TAGAACCCAAAGGAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATCGA301 AGTAATAACGATTAATCACAGAACCCATATTATAATATAGGTTCTTTATTTTATTTTTAG361 AATGAAATTAGGAATGATTATGAAATAGAAAATTCATAATTTTTTTTTAGAATTATTGTG421 AATCTATTCCAATCAAATATTGAGTAATCAAATCCTTCAATTCATTGTTTTCGAGATCTT481 TTAATTTTAAAAAGTGGATTAATCGGACGAGGATAAAGAGAGAGTCCCATTCTACATGTC541 AATACTGACAACAATGAAATTTCTAGTAAAAGGAAAATCCGTCGACTTTATAAGTCGTGA601 GGGTTCAAGTCCCTCTATCCCCAAACCCTCTTTTATTCCC

The sequence highlighted in bold (SEQ ID NO: 10) was detected using themolecular beacon probe shown below (SEQ ID NO: 13). The sequencesunderlined show the sequences to which the primers shown below (SEQ IDNOs: 11 and 12) bind.

IIIa - Internal Control Oligonucleotide Primers (SEQ ID NO: 11)5′-CCTGCTAAGTGGTAACTTCC-3′ (SEQ ID NO: 12) 5′-TGAGTCTCTGCACCTATCCT-3′IIIb - Internal Control Molecular Beacon Probe (SEQ ID NO: 13)5′-CGCACGCAATCCTGAGCCAAATCCCTCGTGCG-3′(Hairpin structure nucleotides italicized) IV - Aspergillus(SEQ ID NO: 14) Sequence detected: AGTTGAACCTTGGGTCTGGCIVa - Aspergillus Oligonucleotide Primers (SEQ ID NO: 15)5′-GGTAATTCCAGCTCCAATAGC-3′ (SEQ ID NO: 16) 5′-GGCCTGCTTTGAACACTCTAA-3′IVb - Aspergillus Molecular Beacon Probe (SEQ ID NO: 17)5′-CGCGATAGTTGAACCTTGGGTCTGGCATCGCG-3′(Hairpin structure nucleotides italicized) V Pneumocystis(SEQ ID NO: 18) Sequence detected: CTAGGATATAGCTGGTTTTCTGCVa - Pneumocystis Oligonucleotide Primers (SEQ ID NO: 19)5′-GCAAAGTACTCAGAAGAATTGTGG-3′ (SEQ ID NO: 20)5′-TCCCTCGAGATATTCAGTGC-3′ Vb - Pneumocystis Molecular Beacon Probe(SEQ ID NO: 21) 5′-CGCAGCCT AGG AT AT AGCTGGTTTTCTGCGCTGCG-3′(Hairpin structure nucleotides italicized) VI - Candida tropicalis(SEQ ID NO: 30) Sequence detected: CCTTTTGGCGAACCCAGGACVIa - Oligonucleotide Primers (SEQ ID NO: 31) 5′-ATTGGAGGGCAAGTCTGGTG-3′(SEQ ID NO: 32) 5′-CCGATCCCTAGTCGGCATAG-3′VIb - Candida tropicalis Molecular Beacon Probe (SEQ ID NO: 33)5′-CGCAGCCCTTTTGGCGAACCCAGGACGCTGCG-3′(Hairpin structure nucleotides italicized) VII - Candida parapsilosis(SEQ ID NO: 34) Sequence detected: TCTGGCTAGCCTTTTTGGCGVIIa - Oligonucleotide Primers - SEQ ID NOs: 31 and 32 aboveVIIb - Candida parapsilosis Molecular Beacon Probe (SEQ ID NO: 35)5′-CGGACGTCTGGCT AGCCTTTTTGGCGCGTCCG-3′(Hairpin structure nucleotides italicized) VIII - Candida albicans(SEQ ID NO: 36) Sequence detected: TTCTGGGTAGCCATTTATGGVIIIa - Oligonucleotide Primers - SEQ ID NOs: 31 and 32 aboveVIIIb - Candida albicans Molecular Beacon Probe (SEQ ID NO: 37)5′-CGGACGTTCTGGGT AGCCATTT ATGGCGTCCG-3′(Hairpin structure nucleotides italicized) IX - Candida glabrata(SEQ ID NO: 38) Sequence detected: GCTAACCCCAAGTCCTTGTGIXa - Oligonucleotide Primers - SEQ ID NOs: 31 and 32 aboveIXb - Candida glabrata Molecular Beacon Probe (SEQ ID NO: 39)5′-CGGACGGCTAACCCCAAGTCCTTGTGCGTCCG-3′(Hairpin structure nucleotides italicized) X - Candida krusei(SEQ ID NO: 40) Sequence detected: TCGGGCGAACCAGGACGATTXa - Oligonucleotide Primers SEQ ID NOs: 31 and 32 above OR(SEQ ID NO: 41) 5′-GCAGTTAAAAAGCTCGTAGTTGAAC-3′ (SEQ ID NO: 42)5′-AAAGGCCTGCTTTGAACACTCT-3′ Xb - Candida krusei Molecular Beacon Probe(SEQ ID NO: 43) 5′-CGCAGCTCGGGCGAACCAGGACGATTGCTGCG-3′(Hairpin structure nucleotides italicized)

Example 1—Melting Curve for Panfungal Molecular Beacon Probes

Reported sequences for the design of primers and molecular beacon probeswere used. Molecular beacon probes and DNA primers (Table 1, I) weredesigned using Beacon Designer 3.0 software (PREMIER Biosoft, Palo Alto,Calif.). The default software parameters were applied for all molecularbeacon probe and primer construction. Molecular beacon probes werelabeled with fluorophores 5-carboxyfluorescein (FAM) at the 5′ end andwith dabcyl at the 3′ end. The molecular beacon probes and primers werepurchased from Biosearch Technologies (Biosearch Technologies, Novato,Calif.). The hybridization properties of the molecular beacon probeswere tested for the full temperature range, 25° C.-95° C., withsingle-stranded target oligonucleotides. Molecular beacon probe-targethybridization was performed with the Stratagene MX4000 MultiplexQuantitative PCR system (Stratagene, La Jolla, Calif.). The “MolecularBeacon Melting Curve” experiment type was chosen in the MX4000 softwarefor data monitoring and analysis.

Each 50 μl hybridization reaction mixture contained I× Stratagene CorePCR buffer, 4 mM MgCl₂, 100 pmol of individual target oligonucleotideand 5 pmol of molecular beacon probe. The thermal conditions ofexperiment comprised heating at 95° C. for 3 minutes and cooling to 80°C. with subsequent cooling down to 25° C. using 112, 30-seconds stepswith a temperature gradient −0.5° C. Fluorescence output for eachindividual reaction was measured at the end of the cooling step. Meltingtemperature (T_(n)) for each molecular beacon-target pair was determinedby MX4000 software as a temperature point corresponding to maximal valueof the first derivative of the fluorescence output −R′(T).

Example 2—Detection of any Fungus (Panfungal Detection)

A real-time amplification assay was carried out using the primers andprobes described in Table I (SEQ ID NOs: 2 to 4). The assay included DNAamplification by the polymerase chain reaction (PCR) with real-timedetection utilizing molecular beacon probes. DNA from multiple fungalspecies were tested, together with negative controls consisting of DNAextracted from a Gram positive and Gram negative bacteria. Speciestested were Aspergillus flavus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus,Candida albicans, Candida dubliniensis, Candida glabrata, Candida keyfr,Candida krusei, Candida lusitaniae, Candida lipolytica, Candidaparapsilosis, Candida rugosa, Candida tropicalis, Cryptococcusneoformans, Saccharomyces cerevisiae, Pneumocystis carinii, Pneumocystisjirovecii, Escherichia coli and Staphylococcus aureus. With theexception of the Pneumocystis samples, which were obtained from clinicalsamples, the DNA used was derived from strains obtained from ATCC (ATCC,Manassas, Va., USA)

The procedure was as follows. Real-time PCR experiments were performedon a Stratagene Mx4000 Multiplex Quantitative PCR System using the“Quantitative PCR (Multiple Standards)” setting. Reagents fromEurogentec (Liege, Belgium) were used for all reactions. Each 50-μI PCRreaction contained 1× Eurogentec reaction buffer, 0.2 μM of molecularbeacon probe (lb, SEQ ID NO: 3), 0.25 μM of each of the primers of Iashown in Table 1 (SEQ ID NOs: 1 and 2), 1.25 U of HotGoldStart Taq DNApolymerase (Eurogentec, Liege, Belgium), 0.4 mM dNTPs, 5 mM MgCl₂between 10 pg and 100 ng of fungal chromosomal DNA. Real-time PCRthermal cycler parameters were: 1 cycle of 10 min at 95° C., 45 cyclesof 30 s at 95° C., 30 s at 50° C. and 30 s at 72° C. The filter gain setof the Mx4000 System was changed to FAM-960. The fluorescence wasmeasured 3 times during the annealing step.

Fluorescence signals coming from Stratagene Mx4000 System during PCRamplification were monitored using Mx4000 software in real time. At theend of each run, the amplification plots data were converted to graphicformat and stored as image files or exported into Microsoft Office Exceland stored as spreadsheet files. Total changes in fluorescence forindividual fluorophores (Rpost-Rpre) were taken as values for analysis.Results were converted to graphic or numerical format and stored asimage or spreadsheet files. A summary of the results is shown in Table2.

TABLE 2 A summary of the results of a real time PCR experiment usingpanfungal primers and molecular beacon probes (Ia and lb (SEQ ID NOs: 2to 4) in Table 1) to detect DNA from a variety of fungal and bacterialorganisms. Realtime PCR Sample Organism Result 1 A. flavus + 2 A.fumigatus + 3 A. japonicus + 4 A. nidulans + 5 A. niger + 6 A. terreus +7 C. albicans + 8 C. dubliniensis + 9 C. glabrata + 10 C.guillermondii + 11 C. keyfr + 12 C. krusei + 13 C. lusitaniae + 14 C.lipolytica + 15 C. parapsilosis + 16 C. rugosa + 17 C. tropicalis + 18C. neoformans + 19 S. cerevisiae + 20 P. carinii + 21 P. jirovecii + 22E. coil − 23 S. aureus − 24 Water −

Example 3—Multiplex Detection of Aspergillus and Candida DNA

A real-time amplification assay was carried out for the detection ofAspergillus, Candida and panfungal (presence of any fungal) DNA usingthe primers and probes described in Table 1, I (SEQ ID NOs: 2 to 4), II(SEQ ID NOs: 6 to 7) and IV (SEQ ID NOs: 15 to 17), together with anassay for the presence of the Internal Control DNA using the primers andprobes described in Table 1, III (SEQ ID NOs: 11 to 13). Fluorophoreswere conjugated to the beacon probes to allow detection. Thefluorophores used are shown in Table 3.

TABLE 3 The fluorophores used to label each molecular beacon probeMolecular Beacon Probe Fluorophore Aspergillus HEX(hexachlorofluorescein phosphoramidite) Candida FAM (carboxyfluorescein)Panfungal Cy ®5 (a registered trade mark of GE Lifesciences. FIG. 11shows the structure of Cy5 mono NHS ester) Internal Control Texas Red ®(registered trade mark of Molecular Probes, Inc. FIG. 12 shows thestructure of Texas Red sulphonyl chloride).

The assay included DNA amplification by the polymerase chain reaction(PCR) with real-time detection utilizing molecular beacon probes. DNAfrom multiple fungal species was tested, together with a DNA-templatefree negative control.

Real-time PCR experiments were performed on Stratagene Mx4000 MultiplexQuantitative PCR System using the “Quantitative PCR (MultipleStandards)” setting. Eurogentec qPCR MasterMix No ROX Kit was used forall reactions. Each 50-μl PCR reaction contained, 1× pPCR MasterMix with4 mM MgCl₂, 20 pmol of each molecular beacon, 10-20 pmol of each primerand up to 100 ng of each DNA or water for the no template control. PCRreactions were performed using the parameters, as follows: 1 cycle of 10min at 95° C., 45 cycles of 30 s at 95° C., 30 s at 50° C. and 30 s at72° C. The fluorescence was measured 30 times during the annealing step.

Fluorescence signals coming from Stratagene Mx4000 System during PCRamplification were monitored using Mx4000 software in real time. At theend of each run, the amplification plots data were converted to graphicformat and stored as image files or exported into Microsoft Office Exceland stored as spreadsheet files. Total changes in fluorescence forindividual fluorophores (Rpost-Rpre) were taken as values for analysis.Results were converted to graphic or numerical format and stored asimage or spreadsheet files. A summary of the results is shown in FIG. 2.

Example 4—Multiplex Detection of Aspergillus and Pneumocytsis DNA

A real-time amplification assay was carried out for the detection ofAspergillus, Pneumocystis (PCP) and panfungal (presence of any fungal)DNA using primers and probes described in Table 1, I (SEQ ID NOs: 2 to4), IV (SEQ ID NOs: 15 to 17) and V (SEQ ID NOs: 19 to 21), togetherwith an assay for the presence of the Internal Control DNA using theprimers and probes described in Table 1, III (SEQ ID NOs: 11 to 13).

Fluorophores were conjugated to the beacon probes to allow detection.The fluorophores used are shown in Table 4.

TABLE 4 The fluorophores used to label each molecular beacon probeBeacon probe Fluorophore Aspergillus HEX (hexachlorofluoresceinphosphoramidite) Pneumocystis FAM (carboxyfluorescein) Panfungal Cy ®5(a registered trade mark of GE Lifesciences. FIG. 11 shows the structureof Cy5 mono NHS ester) Internal Control Texas Red ® (registered trademark of Molecular Probes, Inc. FIG. 12 shows the structure of Texas Redsulphonyl chloride).

The assay included DNA amplification by the polymerase chain reaction(PCR) with real-time detection utilizing molecular beacon probes. DNAfrom multiple fungal species were tested, together with a DNA-templatefree negative control.

Real-time PCR experiments were performed on Stratagene Mx4000 MultiplexQuantitative PCR System using the “Quantitative PCR (MultipleStandards)” setting. Eurogentec qPCR MasterMix No ROX Kit was used forall reactions. Each 50111 PCR reaction contained, 1× pPCR MasterMix with4 mM MgCl₂, 20 pmol of each molecular beacon, 10-20 pmol of each primerand up to 100 ng of each DNA or water for the no template control. PCRreactions were performed using the parameters, as follows: 1 cycle of 10min at 95° C., 45 cycles of 30 s at 95° C., 30 s at 50° C. and 30 s at72° C. The fluorescence was measured 30 times during the annealing step.

Fluorescence signals coming from Stratagene Mx4000 System during PCRamplification were monitored using Mx4000 software in real time. At theend of each run, the amplification plots data were converted to graphicformat and stored as image files or exported into Microsoft Office Exceland stored as spreadsheet files. Total changes in fluorescence forindividual fluorophores (Rpost-Rpre) were taken as values for analysis.Results were converted to graphic or numerical format and stored asimage or spreadsheet files. A summary of the results is shown in FIG. 3.

Example 5—Detection of Fungus in Non-Biological Samples

A real-time amplification assay was carried out for the detection ofpanfungal (presence of any fungal) DNA using primers and probesdescribed in Table 1, I (SEQ ID NOs: 2 to 4). Nine samples of water weretaken from various rooms throughout a Hospital and were tested togetherwith sterilized laboratory water, a positive (extracted Aspergillusgenomic DNA) and negative (sterile PBS) control.

The procedure was as follows. Real-time PCR experiments were performedon a Stratagene Mx4000 Multiplex Quantitative PCR System using the“Quantitative PCR (Multiple Standards)” setting. Reagents fromEurogentec (Liege, Belgium) were used for all reactions. Each 50-μl PCRreaction contained 1× Eurogentec reaction buffer, 0.2 tiM of molecularbeacon probe (lb, SEQ ID NO: 3), 0.25 μM of each of the primers of Iashown in Table 1 (SEQ ID NOs: 1 and 2), 1.25 U of HotGoldStart Taq DNApolymerase (Eurogentec, Liege, Belgium), 0.4 mM dNTPs, 5 mM MgCl₂between 10 pg and 100 ng of fungal chromosomal DNA. Real-time PCRthermal cycler parameters were: 1 cycle of 10 min at 95° C., 45 cyclesof 30 s at 95° C., 30 s at 50° C. and 30 s at 72° C. The filter gain setof the Mx4000 System was changed to FAM-960. The fluorescence wasmeasured 3 times during the annealing step.

Fluorescence signals coming from Stratagene Mx4000 System during PCRamplification were monitored using Mx4000 software in real time. At theend of each run, the amplification plots data were converted to graphicformat and stored as image files or exported into Microsoft Office Exceland stored as spreadsheet files. Total changes in fluorescence forindividual fluorophores (Rpost-Rpre) were taken as values for analysis.Results were converted to graphic or numerical format and stored asimage or spreadsheet files. A summary of the results is shown in Table 5and FIG. 4.

TABLE 5 A summary of the results of a real time PCR experiment usingpanfungal primers and molecular beacon probes (1a and 1b (SEQ ID NOs: 2to 4) in Table 1) to detect panfungal DNA in a variety of Hospital watersamples. Well Threshol Sample Well Assay Type d (dR) Ct (dR) no. Sampletype D4 FAM Unknown 833.626 52 1 Room 16, patient bathroom D5 FAMUnknown 833.626 41.52 2 Room 9, patient bathroom D6 FAM Unknown 833.626No Ct 3 Hepa room 19, pateint bathroom D7 FAM Unknown 833.626 35.78 4Room 2, patient bathroom D8 FAM Unknown 833.626 38.8 5 Proceedure roomout patient area D9 FAM Unknown 833.626 No Ct 6 Main kitchen E4 FAMUnknown 833.626 44.35 7 Stores Basement Sluice E5 FAM Unknown 833.62643.05 8 Ward 1 kitchen E6 FAM Unknown 833.626 58.74 9 May Draper Tea barE7 FAM Unknown 833.626 No Ct control Wythenshawe ERC lab E8 FAM Unknown833.626 33 control positive E9 FAM NTC 833.626 No Ct control negative

Example 6—Detection of Specific Species of Candida

Experiments were performed as discussed above for Examples 2 to 5.

A real-time amplification assay was carried out for the detection ofCandida tropicalis using primers and probes described in Table 1, VI(SEQ ID NOs: 31 to 33). The other samples simultaneously tested in amultiplex reaction were DNA extracted from Candida albicans, Candidakrusei, Candida neoformans, Candida dubliniensis, Candida lusitaniae,Staphylococcus. Cerevisiae, Candida glabrata, Candida parapsilosis,Aspergillus fumigatus, Candida guilliermondii and Candida tropicalis aswell as water. A summary of the results is shown in FIG. 6.

A real-time amplification assay was carried out for the detection ofCandida parapsilosis using primers and probes described in Table 1, VII(SEQ ID NOs: 31, 32 and 35). The samples tested were the same as testedfor Candida tropicalis. A summary of the results is shown in FIG. 7.

A real-time amplification assay was carried out for the detection ofCandida albicans using primers and probes described in Table 1, VIII(SEQ ID NOs: 31, 32 and 37). The samples tested were the same as testedfor Candida tropicalis. A summary of the results is shown in FIG. 8.

A real-time amplification assay was carried out for the detection ofCandida glabrata using primers and probes described in Table 1, IX (SEQID NOs: 31, 32 and 39). The samples tested were the same as tested forCandida tropicalis. A summary of the results is shown in FIG. 9.

A real-time amplification assay was carried out for the detection ofCandida krusei using primers and probes described in Table 1, X (SEQ IDNOs: 31, 32 and 43). The samples tested were the same as tested forCandida tropicalis. A summary of the results is shown in FIG. 10.

It is understood that the present invention is not limited to thepreferred embodiments and examples presented herein, which serve only toillustrate certain aspects of the invention to one of ordinary skill inthe art. Other embodiments of the invention, which would be apparent tothose of ordinary skill, are considered to fall within the scope andspirit of the invention.

Example 7—Use of Four Color Multiplex Molecular Beacon Probes for theDetection of Fungal Pathogens and an Internal Control

A four color multiplex approach for the detection of pathogenic fungiand an internal control was developed for real time quantitative PCRwith molecular beacon probes. The multiplex panel included the detectionof all fungi (panfungal), all Candida species (panCandida), allApspergillus species (panAspergillus) and a novel non-human andnon-fungal internal control DNA sequence (internal control).

To enhance detection sensitivity for the panAspergillus probe, two newprimers were designed (SEQ ID Nos 46 and 47) and new target regions inthe 18S ribosomal gene were also identified that had broad panfungalcoverage. Using these regions, primers, denoted as “v.2” and “v.3,” andmolecular beacons, denoted as “v.2.3” and “v.3.3,” were designed. SeeTables 6 and 7 below.

Two panels of four color multiplex probes were employed using eitherPanfungal v2.3 or v.3.3 plus the internal control, panCandida andpanAspergillus. Each panel was evaluated using a variety of targetgenomic DNA to assess the multiplex specificity.

Real-time PCR experiments were performed on a Stratagene Mx4000Multiplex Quantitative PCR System using the “Quantitative PCR (MultipleStandards)” setting. Eurogentec qPCR MasterMix No ROX Kit was used forall reactions. Each 50 μl PCR reaction contained, 1× pPCR MasterMix with4 mM MgCl₂, 20 pmol of each molecular beacon, 10-20 pmol of each primerand up to 100 ng of each DNA or water for the no template control. PCRreactions were performed as follows: 1 cycle of 10 min at 95° C., 45cycles of 30 sat 95° C., 30 sat 50° C. and 30 sat 72° C. Thefluorescence was measured 30 times during the annealing step.

Real time PCR was performed with the two multiplex panels using a singletemplate to evaluate each panel's specificity. A. fumigatus, C.albicans, C. neoformans, R. oryzae, S. aureus and Z. mays genomic DNAwere used as templates for this assay.

TABLE 6 Molecular beacon probes Molecular 5′ Beacon Reporter 3′ ProbeDye Quencher Oligonucleotide sequence panCandida HEX DABCYLcgcgatGATGATTCATAATAACTTTTCGatcgcg (PanCan) (SEQ ID NO: 8)panAspergillus FAM DABCYL cgcgatAGTTGAACCTTGGGTCTGGCatcgcg (PanAsp)(SEQ ID NO: 17) panfungal Cy5 DABCYL ccgtggCTGCGGCTTAATTTGACTCAccacgg(PanFunv2.3) (SEQ ID NO: 44) panfungal Cy5 DABCYLcgcgacACATCCAAGGAAGGCAGCAGgtcgcg (PanFunv3.3) (SEQ ID NO: 45)Internal Control (IC) Texas Red DABCYL cgcacgCAATCCTGAGCCAAATCCCTcgtgcg

TABLE 7 Real time PCR primers Primer Name Direction 5′-3′ SequencePanCandida sense TAGATAAAAAATCAATGCCTTCGG (SEQ ID NO: 6) (SEQ ID NO: 6)antisense CATGGTAGGCCACTATCCTAC (SEQ ID NO: 7) PanAspergillus senseTAATTCCAGCTCCAATAGCG v.1.3 (SEQ ID NO: 46) antisenseCCAGAAGGAAAGGTCCAGCC (SEQ ID NO: 47) PanFungal v.2 senseAATTGACGGAAGGGCACCAC (SEQ ID NO: 48) antisense TGTCTGGACCTGGTGAGTTT(SEQ ID NO: 49) PanFungal v.3 sense AGAGGGAGCCTGAGAAACGG (SEQ ID NO: 50)antisense CGGCTGCTGGCACCAGACTT (SEQ ID NO: 51) Internal  senseCCTGCTAAGTGGTAACTTCC Control (SEQ ID NO: 11) antisenseTGAGTCTCTGCACCTATCCT (SEQ ID NO: 12)

FIG. 13 provides graphical displays of real-time PCR results frommultiplex panel experiments.

Real-time PCR experiments were performed on a Stratagene Mx4000Multiplex Quantitative PCR System using the “Quantitative PCR (MultipleStandards)” setting. Stratagene Brilliant QPCR MasterMix was used forall reactions. Each 25-μl PCR reaction contained 1×QPCR MasterMix with5.5 mM MgCl2, 5 pmol of each molecular beacon, 10 pmol of each primerand up to 100 ng of each DNA or water for the no template control. PCRreactions were performed as follows: 1 cycle of 10 min at 95° C., 45cycles of 30 s at 95° C., 30 s at 50° C. and 30 s at 72° C. Thefluorescence was measured 3 times during the annealing step.

The two panfungal probes assayed in simplex reactions identified a panelof diverse fungal DNA (n=21) that included Zyogmycetes. Both probes alsodetected signal from water containing Ochronis gallopavum andrespiratory samples from patients which did not grow various humanpathogenic fungi, suggesting increased sensitivity of detection. ThepanAspergillus primers increased the detection sensitivity by at least10-fold when compared to earlier primers in a multiplex primer assaythat contained the PanAsp beacon and all the primers in the multiplex.The panCandida probe produced lower fluorescence than other beacons as aresult of incorrect gain settings on the HEX detection channel of theStratagene instrument. In general, the specific assays in both multiplexpanels effectively detected their respective analytes, confirming thefunctionality of each multiplex panel.

Example 8—Multiplex Detection of Aspergillus and Pnemocytis jirovecii

A real-time amplification assay was designed to simultaneously detectnumerous Aspergillus species, Pneumocystis jirovecii and an internalcontrol. The assay included DNA amplification by the polymerase chainreaction (PCR) with real-time detection utilizing molecular beaconprobes. In particular, the primers and probes described in Tables 8-9were employed using these amplification and detection conditions. Thesetup required a total volume of 25 uL, of which 5 uL was clinicalsample, positive or negative control. The Positive Control molecule wasa recombinant plasmid harboring the Aspergillus and Pneumocystis targetsequences. The microtitre tray was prepared in a PCR hood and run on anAB7500 thermocycler using the following run conditions: a single cycleof 95° C. for 10 min then 95° C. for 30 sec, 60° C. for 1 min and 72° C.for 30 sec, for 40 cycles. The threshold setting was set at 0.2 forAspergillus, at 0.1 for Pneumocystis and IAC.

The Aspergillus DNA template lies within the 18S region, which has 35-90copies per genome. The template is common to all Aspergillus genus andpossesses little overlap with other fungal pathogens. The P. jiroveciiiDNA template lies within the Mitchondrial Large Sub Unit.

TABLE 8 Molecular beacon probes Molecular 5′ Beacon Reporter 3′ ProbeDye Quencher Oligonucleotide sequence Aspergillus FAM DDQ1CGCGATAGTTGAACCTTGGGTCTGGCATCGCG Beacon (SEQ ID NO: 17) Pneumocystis HEXDDQ1 CGCAGCCTAGGATATAGCTGGTTTTCTGCGCTGC Beacon (SEQ ID NO: 21) InternalAtto647N DDQ1 CGCACGCAATCCTGAGCCAAATCCCTCGTGCG Control (SEQ ID NO: 13)Beacon DDQ1 = Deep Dark Quencher 1

TABLE 9 Amplification primers Primer Name Direction 5′-3′ SequenceAspergillus Sense GGTAATTCCAGCTCCAATAGC primer, sense (SEQ ID NO: 15)Aspergillus Antisense GGCCTGCTTTGAACACTCTAA primer, (SEQ ID NO: 16)antisense Pneumocystis Sense GCAAAGfACTCAGAAGAATTGTGG primer, sense(SEQ ID NO: 19) Pneumocystis Antisense TCCCTCGAGATATTCAGTGC primer,(SEQ ID NO: 20) antisense Internal Sense CCTGCTAAGTGGTAACTTCC control(SEQ ID NO: 11) primer, sense Internal Antisense TGAGTCTCTGCACCTATCCTcontrol (SEQ ID NO: 12) primer, antisenseSample Processing

Fungal DNA from clinical respiratory (and in some cases, other) sampleswere obtained from clinical collaborators. The samples had been storedin most instances for a number of years, either as the clinicalspecimens or as DNA. A small number of samples were collectedprospectively. Where DNA had not been extracted from the samples, thiswas achieved using MycXtra™ Fungal DNA Extraction Kit (Myconostica,Manchester, UK) containing resin beads.

The methods used in each centre for microscopy and culture of BAL aresummarized in the table below. The samples from Manchester were taken aspart of a research study with informed consent but processed by standardmicrobiology procedures.

Table 10.

Bronchoalveolar lavage fluid (BAL) Processing Manchester PrimaryInnsbruck Lausanne Leuven CL3 laboratory + processing Class 2 hood Class2 hood Class 2 hood class 1 hood Microscopy All BALs Yes when Only onrequest yes (gram stain only) for fungi requested Volume 1 drop 50-250uL 0.4 mL 1 drop used Centrifuged? 15 000 g/5 min; CytocentrifugationYes, yes resuspend cytocentrifuge Method Calcoflour White GMSFungi-FluorTM gram stain Kit PCP Calcafluor GMS Yes, if urgent, GMSstain method Immy kit (histopathology) Volume 50-250 ul UsedCentrifuged? 15 000 g/5 min; Cytocentrifugation Resuspend in 5 mL FungalYes if req, or All culture yeast/hyphae seen Volume 1-5 mL 5 mL 1-2drops 2-3 drops used Centrifuged? 15 000 g/5 min; 3000 rpm No Yes 3000rpm/5 mins resuspend (2000 g) × 10 min Medium 1 Sabouraud + SabouraudSabouraud + Sabouraud + Cloramphenicol chloramphenicol chloramphenicolMedium 2 Brain heart Infusion Medium 3 Malt exract Plate or Plate SlantSlant slant? Temperature 30 C., 37 C. 30 C. 35-37 C. for 48 hrs 30 C.for 10 days (s) then 30 C.

As can be seen, the method of sample handling varied by centre. Both thevolume of BAL used for standard and Pneumocystis microscopy and fungalculture varied, as well. For example, Innsbruck, Lausanne and Manchesterused a centrifugation step prior to fungal culture, whereas Leuven didnot. These differences may account for some of the differences in yieldobserved. At the Leuven site, 800 μL of sample was used for DNAextraction with the MycXtra™ kit, and there was no initialcentrifugation step to collect and concentrate fungal cells. Onceextracted, nucleic acid from each sample was shipped and stored at −80°C. prior to testing.

Specificity

Table 11 provides the results of cross reactivity testing using avariety of fungi.

TABLE 11 Aspergillus Pneumocystis Organism Channel Channel Aspergillusfumigates 2/2 0/2 Aspergillus niger 10/10 0/9 Aspergillus terreus 6/60/6 Aspergillus nidulans 3/3 0/3 Aspergillusflavus 2/2 0/2 Aspergillusversicolor 2/2 0/2 Aspergillus glaucus 1/1 0/1 Aspergillus tubingensis2/2 0/2 Aspergillus foetidus 2/2 0/2 All Aspergillus 30/30  0/30Penicillium notatum 1/1 0/1 Penicillium spp. 3/3 0/3 All Penicillium 4/40/4 Fusarium solani 1/1 1/I  Fusarium dimerium 1/1 0/1 All Fusarium 2/21/2 Cladosporium spp, 1/1 0/1 A lternaria alternaria 0/1 0/1 Rhizomucorpusillus 0/1 0/1 Scedosporium prolificans 0/1 0/1 Candida albicans 0/50/5 Candida glabrata 0/3 0/3 Candida tropicalis 0/1 0/1 Candida krusei0/3 0/3 Candida parapsilosis 0/5 0/5 Candida sake 0/2 0/2 Candidanovogenesis 0/1 0/1 Candida kefyr 0/2 0/2 Candida rugosa 0/2 0/2 Candidainconspicua 0/2 0/2 Candida humicola 0/1 0/1 All Candida  0/27  0/27Trichosporon cutaneum 1/3 0/3 Trichosporon asahii 0/1 0/1 Trichosporonmucoides 0/1 0/1 All Trichosporon 1/5 0/5 Sporothrix schenkii 1/2 0/2Saccharomyces cerevisiae 0/5 0/5 Cryptococcus neoformans 0/7 0/7Rhodotorula rubrum 0/2 0/2 Blastoschizomyces capitatus 0/2 0/2Geotrichum candidum 0/1 0/1 Viral DNA 0/7 0/7 Bacterial DNA 0/3 0/3Human DNA 0/1 0/1Sensitivity

Analytical Sensitivity for Aspergillus was determined using quantifiedDNA extracted from A. fumigatus A293. FIG. 14 shows that a Limit ofDetection of one genomecopy can be obtained using standard methodology(CLSI EP17-A—“Protocols for Determination of Limits of Detection andLimits of Quantitation”).

Analytical Sensitivity for Pneumocystis was determined using quantifiedrecombinant plasmid DNA. FIG. 15 shows that a Limit of Detection of sixtarget copies can be obtained using standard methodology (CLSIEP17-A—“Protocols for Determination of Limits of Detection and Limits ofQuantitation”). The target is a multicopy gene, but the number of copieswithin the genome, and therefore the per-organism detection limit isunknown.

Table 12 provides the sensitivity and limit of detection for theseprimers and probes.

TABLE 12 Limit of Detection (Ct) Target number equivalent Aspergillusfumigatus 38.6 34 Pneumocystis jirovecii 38.5 6

The value for the Aspergillus target was determined using the A293strain of Aspergillus fumigatus from which the genome has been fullysequenced. It is known there are 37 copies of the target within thegenome, determined by optical mapping and thus 34 target copiesrepresent approximately 1 genome copy.

The value for the Pneumocystis target was determined using a recombinantDNA plasmid harbouring the target sequence. As the Pneumocystis targetsequence is a mitochondrial target, there will be numerous copies percell, but it not known how many. For comparison there are 13mitochondrial genomes per A. fumigatus nuclear genome (in AF293) and anunknown number of subunit repeat sequences per mitochondrion inPneumocystis jirovecii.

Internal Amplification Control

There was some preliminary evidence of the introduction of a low-levelinhibitor into the reaction from the extraction process. This wasovercome with a smaller eluate volume in the FXG assay. Therefore inorder to assess the effect of altering the amount of sample included inthe reaction, the Internal Amplification Control values of the sampleswere examined and compared. The reproducibility of the internalamplification control was determined inl 32 assays conducted in 6experiments (table below). The cut-off was taken as +/3 standarddeviations from the mean (Ct=32-36.2).

TABLE 13 Range Mean SD 3 × SD Min Max 34.58 0.54 1.61 33.0 36.2

In the tests of the clinical samples where 5)11_, of sample was added tothe reaction, only 26 samples of 399 (5.8%) were out of range, 2 with alow Ct value and 24 with an elevated Ct value indication PCR inhibition.13 of these showed complete inhibition of PCR (recorded as a Ct of 41for the purposes of analysis). The mean Ct value for the 399 samplesassayed was 34.68+/−1.42.

In the tests where 2 μL of sample was added to the reaction, only 2 ofthe Internal Amplification Control results were outside of the allowedrange. In both cases the Ct value was lower than the range (i.e. not dueto inhibition). No sample demonstrating inhibition was identified withthe 2 μL test.

Both samples with low Ct values, consistent with an accelerated PCRreaction, were from Lausanne, and this result was seen in both 2 μL and5 μL reactions. The nature of this accelerated reaction was notdetermined.

Overall, the 50 μL volume was selected for further analysis, based onthe demonstration of inhibition in clinical samples and inspection ofthe clinical results and their correlation with culture and microscopy.

Pneumocystis Results

The results were analyzed as follows: (i) samples which had an internalamplification control (IAC) out of range were highlighted and removed;(ii) only those samples with microscopy results which were appropriatefor the detection of P. jiroveciii and/or those in whom the clinicaldiagnosis of Pneumocystis pneumonia was diagnosed were selected; (iii)PCR positive results were sorted by Ct value to assess lowestappropriate cut-off for positivity; and (iv) 2×2 table of results wereconstructed using microscopy (+2 clinically diagnosed cases) todetermine the sensitivity, specificity, positive (PPV) and negativepredictive (NPV) values. A secondary analysis of HIV/AIDS patients alsowas conducted to evaluate the cutoff value in these differentpopulations.

A total of 190 results were analysed: 42 from patients with AIDS or HIVinfection and 148 from other patients. The appropriate cutoff forpositivity was determined to be at the limit of detection of the assayi.e. a Ct value of 38.5. Using this cutoff the results are shown in thefollowing tables:

TABLE 14 PCP AIDS and non-AIDS FXG PCR+ FXG PCR− total % Pneumocystis 672 69 97.10 Sensitivity microscopy+ Pneumocystis 16 105 121 86.78Specificity microscopy− Total 83 107 190 80.72 98.13 PPV NPV

Ct values ranged from 23.55 to the limit of detection (38.5) indicativeof a wide range of organism loads. It can be seen that the sensitivityof 97.1% is a good result with only 2 samples positive by microscopy notdetected. The NPV is very high (98.13) consistent with a highlysensitive test. The specificity was not as high, as 16 samples showedpositive results, but were microscopy negative and no clinical diagnosisof Pneumocystis pneumonia was made. This is consistent with thepublished literature indicating a higher sensitivity of PCR assayscompared with microscopy, and with missed clinical diagnoses. One of the2 samples which was negative was from a non-AIDS patient and the BAL wasnot centrifuged.

The data were separated into AIDS/HIV results and all others for furtheranalysis.

TABLE 15 PCP AIDS/HIV FXG PCR+ FXG PCR− Pneumocystis 36 1 37 97.30Sensitivity microscopy+ Pneumocystis 1 4 5 80.00 Specificity microscopy−Total 37 5 42 97.30 80.00 PPV NPV

The performance of the test in AIDS patients was excellent, and higherCt values were obtained in this group (24.57-34.93). The calculationsfor specificity and NPV are limited by only having 4 true negativesamples for testing.

TABLE 16 PCP non-AIDS/H1V FXG PCR+ FXG PCR− Pneumocystis 31 1 32 96.88Sensitivity microscopy+ Pneumocystis 15 101 116 87.07 Specificitymicroscopy− Total 48 100 148 67.39 99.02 PPV NPV

In the non-AIDS patients a larger number of true negative samples wereobtained, and the Ct range of positive results was greater(23.55-37.55). The sensitivity is high at 96.88, and the NPV is alsohigh at 99.02.

The proposed interpretation criteria in the IFU will enable positiveresults to be reported, even if inhibition is detected (ie a raised IACCt value). These results were excluded from the tables above. Six P.jiroveciii results fall into this category, and in 5 cases themicroscopy was positive (Ct values of 21.06, 22.24, 25.18, 25.26, 33.53and 35.91). The Ct values in these samples containing an inhibitor weregenerally low, and only the highest (35.91) was microscopy negative.

Aspergillus Results

The analysis steps were as follows: (i) results which had an internalamplification control (IAC) out of range were highlighted and removed,(ii) sample types were grouped into BAL centrifuged or not, otherrespiratory specimens (including sputum) and tissue/biopsy samples,(iii) only those results with positive culture results (respiratorysamples) or culture or histology positive for tissue were selected, (iv)PCR positive results were sorted by Ct value to assess lowestappropriate cut-off for positivity, (v) 2×2 tables of results wereconstructed using culture results or histology positive samples todetermine the sensitivity, specificity, positive (PPV) and negativepredictive (NPV) values and (vi) results inspected to determine anypossible impact of prior antifungal therapy on sensitivity.

The cut-off for positivity was determined by inspection of the data andwas determined to be at the limit of detection (Ct 38.6). This cut-offwas reinforced by examination of the 15 samples from Manchester fromnormal controls in which 2 has Ct values of 38.82 and 38.4. Very lowlevels of Aspergillus spp. are found in normal lungs and so a cut-off atthis level is appropriate to detect a pathogen, which is found in smallquantities even in severely ill patients. In fact the sample with a Ctvalue of 38.4 grew Penicillium spp. and another sample grew A.fumigatus, but was negative by the test assay. These data are consistentwith very low levels of fungi in the lungs of normal people and indicatethat the assay is operating at this level of detection.

Based on the IAC cut-off, 10 BAL samples (5 centrifuged and 5uncentrifuged), 5 other respiratory samples and no tissue/biopsy sampleswere excluded from analysis because the IAC was out of range.

The results of the BAL samples centrifuged and non-centrifuged are shownbelow. Although there are slightly fewer samples and fewer positivesamples in the uncentrifuged group, the impact of centrifugation as apreliminary step in extraction appears clear from these data. Thesensitivity falls from 73% to 29%, although the specificity isessentially unchanged. On the basis of this analysis, only centrifugedBAL samples were included in the overall analysis. Centrifugation couldaccount for for the difference, but other factors could be relevant,including the patient population (large proportion of ICU patients),processing differences and storage factors.

TABLE 17 BAL only (centrifuged) FXG PCR+ FXG PCR− Aspergillus 19 8 2670.37 Sensitivity culture +ve Aspergillus 4 92 97 95.83 Specificityculture −ye 23 100 123 82.61 92.00 PPV NPV

TABLE 18 Uncentrifuged BAL samples FXG PCR+ FXG PCR− Aspergillus 5 12 1729.41 Sensitivity culture+ Aspergillus 3 75 77 96.15 Specificityculture− 8 87 95 62.50 86.21 PPV NPV

The other respiratory samples tested included sputum, bronchialaspirations, endotracheal aspirations, lung aspirations, one pulmonarywound drainage and one nasal secretion. Most of the sputum samples werefrom cystic fibrosis patients. The volumes processed varied, and thereis no centrifugation step in the processing of these usually smallsamples. The results are shown below for these 75 samples.

TABLE 19 Sputum and non-BAL respiratory samples FXG PCR+ FXG PCR−Aspergillus 15 4 19 78.95 Sensitivity culture+ Aspergillus 7 49 56 87.50Specificity culture− 22 53 75 68.18 92.45 PPV NPV

Compared with the centrifuged BAL samples, the sensitivity is similar at79% versus 73%. Proportionately there slightly more assay positive,culture negative results with other respiratory samples than with BAL,leading to a slightly lower specificity 88% versus 96%, and lower PPV(68% versus 83%). These differences probably reflect many factors, suchas sample volume and antifungal treatment.

TABLE 20 All respiratory samples together (centrifuged BALs) FXG PCR+FXG PCR− Aspergillus 34 12 45 73.91 Sensitivity culture+ Aspergillus 11142 153 92.76 Specificity culture− 45 153 198 75.56 92.16 PPV NPV

Of the 198 respiratory samples analysed, 34 were both culture positiveand assay positive, 142 were true negatives, with 11 false positive FXGPCR and 12 FXG PCR false negatives. This yields sensitivity and PPVvalues of 73.9% and 75.6% respectively and specificity and NPV values of92.8% and 92.2% respectively. Of note, 3 BAL samples were culturepositive for Penicillium spp., which is not distinguished fromAspergillus spp. by the assay. If these are recorded as true positives,the sensitivity rises to 75.5%, the specificity to 94.6% and the PPV andNPV to 82.2% and 92.2% respectively. Also of note two patients withzygomycosis caused by Rhizopus spp. were not detected by the assay, norwere eight samples from patients colonized by Candida albicans, nor onecolonized by three species of Paecilomyces and Candida tropicalis. Sixmicroscopy positive samples for non-septate hyphae (but culturenegative) were negative in the assay.

There were 15 samples excluded from the analysis because the IAC was outof range. The proposed interpretation criteria in the IFU will allowpositive results to be reported, even if inhibition is detected (i.e. araised IAC Ct value). Among these 15 samples, 13 were both culture andassay negative and two were culture positive and assay negative.Therefore, it is not possible to confirm with this dataset that thisinterpretation is correct, and it will need confirmation in futurestudies.

There were 31 fresh lung tissue samples processed by the MycXtra™extraction method (preceeded by proteinase K digestion) from Innsbruck.These were selected because they were generally positive samples from atrisk patients, and no controls or negatives were processed alongside.Two analyses were done: one compared FXG PCR results with septate hyphae(all Aspergilli causing tissue invasive disease have septate hyphae, asdo other less common invasive fungi such as Scedosporium spp. Fusariumspp., Penicillium spp. and others) and another compared with culture ofAspergillus spp. Both tables are shown below. Cultures were positive forA. fumigatus and A. terreus.

TABLE 21 Septate hyphae only FXG PCR+ FXG PCR− Histology+ 14 9 23 60.87Sensitivity Histology− 0 7 7 100 Specificity 14 16 30 100 43.75 PPV NPV

TABLE 22 Aspergillus culture +ve FXG PCR+ FXG PCR− Aspergillus 12 3 1580.00 Sensitivity culture+ Aspergillus 2 14 16 87.5 Specificity culture−14 17 31 85.71 82.35 PPV NPV

The data show that the culture performance is better than the histologyperformance, principally because there are six more assay positiveresults compared to culture than to histology. This suggests, but doesnot confirm, that some septate hyphae seen are not Aspergilli. Overallthe 100% PPV for the assay compared with histology is helpfulclinically, combined with culture results, and the high sensitivity(80%) compared with culture is acceptable.

The vast majority of positive cultures were A. fumigates with ratherfewer A. terreus positives and one A. flavus positive. The assaydetected these three species, the most common pathogenic species (>98%).One A. niger and one A. glaucus isolate were cultured from samples andboth were negative in the assay. In both cases they were mixed positivecultures, (C. albicans and Rhizopus oryzae). Neither was implicated indisease.

The real-time detection assay performed well for the rapid detection ofPneumocystis jirovecii and Aspergillus spp. in a large retrospectivemulticentre study. The data are consistent with high efficiencyextraction of both fungal genera and detection limits of <10 fungalcells. Pneumocystis is an alveolar pathogen and is therefore present inlarge amounts in disease in the bronchi and small airways. Aspergilliusually cause parenchymal disease (invasive aspergillosis) in whichsmall numbers of fungal cells are present in the airways, but also maycolonise and infect the large airways, with large numbers of fungalcells present. The clinical performance data are consistent with thesedifferent pathologies, with a higher sensitivity demonstrable for P.jirovecii than Aspergillus. The specificity of the Aspergillus componentof the assay is not absolute, and so additional fungi are detected.These are primarily fungi with a similar antifungal susceptibilityprofile to Aspergillus, with the single exception of Fusarium spp. whichis not susceptible to the echinocandins or itraconazole. Test failuredue to PCR inhibition was infrequent, and of uncertain cause, as theMycXtra™ kit removes many potential inhibitors, including inhaled drugs.For Pneumocystis (and by extension to Aspergillus) a positive result canbe safely, even with an inhibitor present, whereas a negative onecannot. Additional data on a small number of lung biopsy specimens showsgood performance, especially when compared to culture.

We claim:
 1. A method for detecting the presence or absence of anyspecies of a fungus belonging to the genus Candida in a sample,comprising: i. obtaining a sample suspected of containing fungal nucleicacid from the genus Candida, and ii. testing for the presence or absencein the sample of an identifying region of fungal nucleic acid comprisingSEQ ID NO: 5, the complement or transcript thereof, or a sequence having80% or more sequence identity with SEQ ID NO: 5, the complement ortranscript thereof, said identifying region being present in species ofthe genus Candida but not those of the genus of Aspergillus orPneumocystis, wherein the testing includes contacting the sample with amolecular beacon probe comprising SEQ ID NO: 8, and wherein the presenceof said identifying region of fungal nucleic acid indicates the presenceof any species of a fungus belonging to the genus Candida in the sample,and the absence of said identifying region of fungal nucleic acid in thesample indicates the absence of any species of a fungus belonging to thegenus Candida in the sample.
 2. The method of claim 1 further comprisingamplifying the identifying region of fungal nucleic acid by contactingthe sample with a pair of primers including SEQ ID NO: 6 and SEQ ID NO:7.
 3. The method of claim 2 in which the amplifying step is carried outin the presence of one or more internal PCR amplification controls. 4.The method of claim 3 in which the one or more internal PCRamplification controls comprise a non-fungal sequence.
 5. The method ofclaim 3 in which the amplifying step is carried out in the presence of acloned or synthesized tRNA-LEU intron region, which is then added to theamplification mixture in a predetermined amount to rule out the presenceof inhibitors or other defective amplification steps.
 6. The method ofclaim 5 in which the tRNA-LEU intro region comprises a portion of theMaize (Zea mays) tRNA-LEU intron region.
 7. The method of claim 5 inwhich the tRNA-LEU intron region includes SEQ ID NO:
 9. 8. The method ofclaim 7 further comprising detecting the presence of a nucleic acidinclude SEQ ID NO:
 10. 9. The method of claim 8 in which the detectingstep comprises contacting the sample with a pair of oligonucleotideprimers including SEQ ID NO: 11 and SEQ ID NO: 12 and a molecular beaconprobe including SEQ ID NO:
 13. 10. The method of claim 1 in which thenucleic acid comprises DNA.
 11. The method of claim 1 in which thenucleic acid comprise RNA.
 12. The method of claim 1 in which the sampleis obtained from a biological source.
 13. The method of claim 12 inwhich the sample comprises a biological fluid, tissue, or combinationthereof.
 14. The method of claim 12 in which the sample is a biologicalsample obtained from a non-biological source.
 15. The method of claim 14in which the non-biological source comprises a piece of a vehicle,watercraft, aircraft, building, or dwelling.
 16. The method of claim 1in which the source is obtained from an environment.
 17. The method ofclaim 16 in which the sample comprises an air sample, a water sample, asoil sample, or combinations thereof.
 18. The method of claim 1 in whichthe testing step further comprises contacting the sample with anoligonucleotide probe comprising a nucleic acid capable of hybridizingto at least one universal region of fungal nucleic acid under stringentconditions.
 19. The method of claim 18 in which the probe includes adetectable label.
 20. The method of claim 1, wherein said method is usedfor the diagnosis of a fungal infection belonging to the genus Candidain a patient, and the sample is from said patient.